Calibration procedure for investigating biological systems

ABSTRACT

Use of calibrant in extraction phase is described for quantification of components of interest in samples in laboratory application as well as in on-site monitoring. This approach is particularly useful for in-vivo investigation of living systems.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/506,827, filed Sep. 7, 2004, derived from InternationalPatent Application PCT/CA2003/0000311. Further, this application isentitled to the benefit of and claims priority to U.S. PatentApplication 60/364,214 filed Mar. 11, 2002; U.S. Patent Application60/393,309 filed Jul. 3, 2002; U.S. Patent Application 60/421,001, filedOct. 25, 2002; U.S. Patent Application 60/421,510 filed Oct. 28, 2002;and U.S. Patent Application 60/427,833 filed Nov. 21, 2002.Additionally, this application is entitled to the benefit of and claimspriority to U.S. Patent Application 60/604,631 filed Aug. 27, 2004. Theentirety of each document is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to calibration methods forquantifying or identifying components of interest in a biologicalsystem, such as in an animal.

BACKGROUND OF THE INVENTION

Presently, if one wants to accurately assess the concentrations ofchemicals or drugs inside a living animal a sample of the blood ortissue to be studied is removed from the animal and taken to ananalytical laboratory to have the chemicals of interest extracted andquantified. Typically a first step is a pre-treatment of the sample toconvert it to a form more suitable for chemical extraction. In the caseof blood this may be by the removal of blood cells and/or some bloodcomponents by the preparation of serum or plasma. In the case of atissue sample this may be by many processes including freezing,grinding, homogenizing, enzyme treatment (eg. protease or cellulase) orhydrolysis. Subsequently chemicals of interest are extracted andconcentrated from the processed sample. For example serum samples may besubjected to liquid-liquid extraction, solid phase extraction or proteinprecipitation followed by drying and reconstitution in an injectionsolvent. A portion of the injection solvent is introduced to ananalytical instrument for chromatographic separation and quantificationof the components. This method produces accurate results with highspecificity for the compound of interest, but is time consuming andlabour intensive. Also, because of the large number of steps in theprocess there is a significant chance of errors in sample preparationimpacting the results. This method has good sensitivity and selectivityand accuracy for the target compounds but is limited in that thechemical balance the chemicals exist in inside the animal is disruptedduring sampling. In many cases this disruption reduces the value of theresults obtained, and in some cases makes this technique inappropriatefor the analysis. Where the blood volume removed is a high proportion ofthe total blood volume of the animal, as is commonly the case when miceare used, the death of the animal results. This means that a differentanimal must be used for each data point and each repeat. By eliminatingthe need for a blood draw in this case, fewer animals would be requiredfor testing and a significant improvement in inter-animal variation inthe results would be achieved.

Alternatively biosensors have been developed for some applications inanalysis of chemical concentrations inside animals. In this case adevice consisting of a specific sensing element with associatedtransducer is implanted and produces a signal collected by an electronicdata logger that is proportional to the chemicals to which the sensorresponds. The main limitations of this type of device are that theynormally respond to a spectrum of chemicals rather than havingspecificity for only one chemical. Of the spectrum of chemicals to whichthe sensor responds, some produce a greater and some a lesser response.Sensors are also susceptible to interferences where another chemicalpresent in a system interferes with the response produced by the targetchemicals. For these reasons biosensors are normally limited in terms ofaccuracy and precision. Finally biosensors are typically not assensitive to low chemical concentrations as state of the art stand alonedetectors such as mass spectrometers that are used in the abovementioned conventional analysis techniques and in solid phasemicroextraction. A strength of this technology is that the chemicalbalance in the system under study is not disturbed.

The in vivo procedure described here is a significant departure fromconventional ‘sampling’ techniques, where a portion of the system understudy is removed from its natural environment and the compounds ofinterest extracted and analyzed in a laboratory environment. There aretwo main motivations for exploring these types of configurations.

The first is the desire to study chemical processes in association withthe normal biochemical milieu of a living system, and the second is thelack of availability or impracticality frequently associated with sizeof removing suitable samples for study from the living system. Newerapproaches that extend the applicability of conventional SPMEtechnology, where an externally coated extraction phase on a micro fibreis used, seem to be logical targets for the development of such tools.As with any microextraction, because compounds of interest are notexhaustively removed from the investigated system, conditions can bedevised where only a small proportion of the total compounds and none ofthe matrix are removed, thus avoiding a disturbance of the normalbalance of chemical components. This could have a benefit in thenon-destructive analysis of very small tissue sites or samples. Finallybecause extracted chemicals are separated chromatographically andquantified by highly sensitive analytical instruments, high accuracy,sensitivity and selectivity are achieved.

With the current commercially available SPME devices a stationaryextraction polymer is coated onto a fused silica fibre. The coatedportion of the fibre is typically 1 cm long and coatings have variousthicknesses. The fibre is mounted into a stainless steel support tubeand housed in a syringe-like device for ease of use. Extractions areperformed by exposing the extraction polymer to a sample for apre-determined time to allow sample components to come into equilibriumwith the extraction phase. After extraction the fibre is removed to ananalytical instrument (typically a gas or liquid chromatograph) whereextracted components are desorbed and analysed. The amount of acomponent extracted is proportional to its concentration in the sample(J. Pawliszyn “Method and Device for Solid Phase Microextraction andDesorption”, U.S. Pat. No. 5,691,206.).

To date commercial SPME devices have been used in some applications ofdirect analysis of living systems. For example they have been appliedfor the analysis of airborne pheromones and semiochemicals used inchemical communications by insects (Moneti, G.; Dani, F. R.; Pieraccini,G. T. S. Rapid Commun. Mass Spectrom. 1997, 11, 857-862.), (Frerot, B.;Malosse, C.; Cain, A. H. J. High Resolut. Chromatogr. 1997, 20,340-342.) and frogs (Smith, B. P.; Zini, C. A.; Pawliszyn, J.; Tyler, M.J.; Hayasaka, Y.; Williams, B.; Caramao, E. B. Chemistry and Ecology2000, 17, 215-225.) respectively. In these cases the living animals werenon-invasively monitored over time by assessing the chemicalconcentrations in the air around the animal, providing a convenientmeans to study complicated dynamic processes without interference.

The current commercial devices do, however, have some limitations for invivo analysis inside a living animal. Firstly, the application tochemical analysis inside animals requires greater robustness in both theextraction phase and the supporting fibre core. In addition, most of theextraction phases currently available are better suited for morevolatile and less polar compounds. Only one phase is suitable for liquidchromatography (LC) applications (carbowax/templated resin). Analytes ofinterest that typically circulate in living systems are less volatileand more polar and require LC analysis, so new or modified extractionphases are indicated. The overall dimension of the current device istypically too large for direct in vivo analysis and for directinterfacing to microanalytical systems, the time required for the LCextraction phase to come into equilibrium with chemicals in a sample isrelatively long (typically 1 hr or more in a well-stirred sample) andanalysis is sensitive to degree of convection in the sample. Also thepresent SPME devices cannot be conveniently coupled to positioningdevices necessary for in-vivo investigation at a well-defined part ofthe living system.

It is, therefore, desirable to provide a method and a device that allowsminimally invasive sampling, quantification or analysis of a biologicalsystem

SUMMARY OF THE INVENTION

It is an object of the present invention to obviate or mitigate at leastone disadvantage of previous devices and methods for evaluatingcomponents of interest in biological systems.

According to an aspect of the invention there is provided a method ofdetermining the concentration of a component in a sample. The methodcomprising steps of: adding a calibrant to extraction phase prior tocontact of said extraction phase with said sample, making said contactbetween said extraction phase and said sample for predeterminedextraction time, terminating said contact and determining saidconcentration of said component using amounts of said calibrant and saidcomponent present in said extraction phase after said contact.

The invention further provides a membrane extraction method ofdetermining concentration of a component in a sample. The methodcomprising the steps of: adding a calibrant to a stripping fluid priorto contact of said stripping fluid with a membrane, making contactbetween said membrane and said sample on one side of said membrane andbetween said membrane and said stripping fluid on the other side of saidmembrane, maintaining said contact to allow said membrane extraction tooccur, introducing said stripping phase into an analytical instrumentand determining said concentration of said component in said sampleusing measured amounts of said calibrant and said component in saidstripping phase.

Additionally, the invention provides a method of delivering a compoundinto investigated system. The method comprising adding a well definedamounts of said compound into coating of a fiber prior to exposure ofsaid fiber to said investigated system.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 shows a general schematic of device design according to anembodiment of the invention.

FIG. 2 illustrates the use of a medical catheter to position deviceaccurately within a vein.

FIG. 3 shows a schematic of the polypyrrole polymerization reaction.

FIG. 4 is a schematic of a catheter with multiple coated fibres.

FIG. 5 shows a schematic of housing and device for soft tissue sampling.

FIG. 6 illustrates operation of housing and device for soft tissuesampling.

FIG. 7 shows a schematic of use of soft tissue sampling housing toposition device for sampling with x-y-z stage.

FIG. 8 illustrates a device according to an embodiment of the inventionwith hollow fibre with inner coated surface with catheter positioningdevice.

FIG. 9 illustrates a device according to an embodiment of the inventionwith hollow fibre sealed at one end with flexible extraction phase.

FIG. 10 shows a chromatogram comparing diazepam extraction from fibrewith polypyrrole only versus fibre with anti-diazepam antibody entrappedin polypyrrole.

FIG. 11 illustrates selective extraction of diazepam using anti-diazepamantibodies immobilized on surface.

FIG. 12 illustrates calibration in whole blood used to calibrate deviceresponse.

FIG. 13 illustrates an example chromatograph obtained after LC/MS/MSquantification of device extraction from plasma.

FIG. 14 shows a cartridge holding a fibre.

FIG. 15 is a schematic of batch process for parallel extraction andmultiple MALDI desorption with positioning devices at both laser sourceand desorption ends of fibres.

FIG. 16 is a schematic of the inventive device used with nanospraynebulizer and ESI MS.

FIG. 17 shows a schematic of fibre/MALDI-IMS system according to theinvention.

FIG. 18 illustrates an exemplary mass spectrum obtained from afibre/MALDI-IMS system according to the invention

FIG. 19 is a schematic of a fibre/MALDI source.

FIG. 20 shows extraction response versus time for standard devices.

FIG. 21 shows extraction response versus time for pre-conditioneddevices.

FIG. 22 provides a comparison of calibration in buffer and plasma,demonstration of linear response limit.

FIG. 23 illustrates an exemplary pharmacokinetic profile of diazepam.

FIG. 24 illustrates an exemplary pharmacokinetic profile of diazepam,with an expanded y-axis.

FIG. 25 illustrates and exemplary pharmacokinetic profile ofnordiazepam.

FIG. 26 illustrates an exemplary pharmacokinetic profile of oxazepam.

FIG. 27 shows an ion mobility spectrum obtained by matrix spray methodat 0.05 mg/mL.

FIG. 28 shows isotropy of absorption of toluene (component) anddesorption of deuterated toluene (calibrant) in SPME using PDMS coatedfibre.

FIG. 29 shows validation of the calibration of uptake of component(fluorine) by use of desorption of calibrants (benzene, toluene,ethylbenzene and xylene).

FIG. 30 calibration of the uptake of component (fluorine) at variousorientation of PDMS coated fibre by use of desorption of calibrant(xylene).

FIG. 31 shows isotropy of absorption of pyrene (component) anddesorption of deuterated pyrene (calibrant) for long exposure of PDMSrod.

FIG. 32 illustrates concentration profiles in membrane extraction.

FIG. 33 shows the impact of flow rate of the sample on mass transfercoefficient of p-xylene (component) and o-xylene (calibrant) and masstransfer coefficient ratio between p-xylene and o-xylene.

FIG. 34 shows the impact of temperature of the sample on mass transfercoefficient of p-xylene (component) and o-xylene (calibrant) and masstransfer coefficient ratio between p-xylene and o-xylene.

FIG. 35 illustrates Diazepam extraction at an intermediate to low flowrate of 50 mL/min.

FIG. 36 shows a multiwell plate for performing parallel SPME fibreassemblies sealing operations.

FIG. 37 shows a holding cover for a multiwell plate for SPME fibreassemblies.

FIG. 38 shows an exposed fibre in a holding cover placed in a well of amultiwell plate.

FIG. 39 shows a fibre in a holding cover retracted into a needle andsealed.

FIG. 40 illustrates a pharmacokinetic profile for Diazepam.

FIG. 41 illustrates a pharmacokinetic profile for Nordiazepam.

FIG. 42 illustrates a pharmacokinetic profile for Oxazepam.

DETAILED DESCRIPTION

The invention relates to a method and device based on coated fibre,optionally in combination with a positioning device, or separation anddetection technologies particularly useful for in vivo studies ofcompounds of interest (identities and concentrations) in animals, orparts of animals.

According to an aspect of the invention, a method of determining theconcentration of a component in a sample is provided. The extractionphase contains calibrant. The extraction phase is placed in contact withthe sample allowing extraction of the component of the sample into theextraction phase. At the same time the calibrant present in theextraction phase is being extracted into the matrix. After the contactbetween the sample matrix and the extraction phase is terminated theamount of the calibrant remaining in the extraction phase and the amountof the component extracted can be used to calculate the concentration ofthe component.

The extraction phase or portion of the extraction phase can be placed inanalytical instrument for determination of the amounts of the amount ofthe calibrant remaining in the extraction phase and the amount of thecomponent extracted during the contact of the extraction phase with thesample. Optionally determination process in the analytical instrumentcan include step of desorption of the calibrant and the component intoorganic solvent contained in wells of a multiwell plate to facilitateparallel desorption step resulting in increase of the throughput ofanalysis.

There are at least two options for length of predetermined extractiontimes. The extraction time can be equal or longer compared to theequilibration time between the extraction phase and the sample allowingfull equilibration of the component between extraction phase and sample.In that case the calibration can be performed based on ratio ofappropriate sample matrix—extraction phase distribution coefficientscorresponding to the calibrant and the component.

As another option, the extraction time can be shorter compared to theequilibration time. In that case the amount of component extractedcorresponds to the rate of mass transfer between the sample matrix andthe extraction phase and the amount of calibrant remaining in theextraction phase to the rate of mass transfer between the extractionphase and the sample matrix. The calibration is performed by using theratio of the corresponding rates.

The calibrant in the extraction phase method is particularly powerfulfor microextraction approach where the small volumes of the extractionphase compared to the volume of the sample matrix are used resulted insubstantial amount of calibrant beeing transported to the sample matrix.When applying this approach to analysis of biological samples in vitroor in-vivo the preferred volume of the extraction phase is less then onemililiter.

The extraction phase can be either gas as in static or dynamic headspacetechniques, or liquid as in liquid extraction or alternatively solid asin sorbent extraction.

Polymer can be used as extraction phase. Polymer can be liquid aspoly(dimethylsiloxane) (PDMS) or poly(ethylene glycol) (PEG) or it canbe solid, for example poly(divinylbenzene) (DVB).

The extraction phase can be made in different shapes to facilitateconvenience of use or increase mass transfer between the extractionphase and the sample matrix. Rods are easy to handle, but flat thinmembranes or coatings have high surface area facilitating rapid masstransfer between the sample matrix and the extraction phase. The coatingconfiguration benefits from the strength of the support.

Fiber geometry and in particularly small coated fiber configuration isvery useful to deliver calibrant to the sample and transfer theextracted component and remaining calibrant from the sample to analysisinstrument. Coated micro fibers are of particular use when samplingliving systems including animals since minimum damage during samplingprocedure occurs.

Tube geometry is another useful format of a support, which facilitatesextraction during drawing the sample through the tube. The tube could bein a form of a neddle or a cartridge.

As another option, the extraction phase can be sealed in the speciallydesigned cartridge to avoid loss of calibrant prior to contact with thesample matrix or after the contact has been terminated to avoid loss ofboth calibrant and the component.

There are at least two options for the level of calibrant release fromthe extraction phase during contact with sample matrix. The calibrantcan be fully retained during the contact. In that case the calibrant isstrongly bound by the extraction phase and only released into analyticalinstrument during desorption. It can be used to calibrate response ofthe instrument or the concentration when the calibrant loadingconditions are identical to the extraction conditions of the compound.

The other option is when the calibrant is partially released from theextraction phase during the contact with sample. In this case thecalibration can be either based on the ratio of correspondingdistribution constants when equilibrium is reached or the ratio ofcorresponding mass transfer coefficients for the pre-equilibriumconditions. The equilibrium case is of particular use when performingin-vitro extractions and the volume of sample is small. In that case theamount of component extracted is maximum resulting in optimumsensitivity. The pre-equilibrium case is very useful for on-site,including in-vivo extractions.

Further, the process of the extraction can be automated including stepof addition of the calibrant to the extraction phase to increasethroughput of the extraction and analysis. For example, the coated fiberlocated in a needle can be automatically exposed first to a vialcontaining calibrant prior to contact with the sample placed in anothervial.

The calibrant is chosen to have similar physicochemical propertiescompared to the component, including the distribution constant and therate of mass transfer between the extraction phase and the matrix toproperly perform calibration. The best choice for calibrant is use ofisotopically labelled analogue of the target component.

According to an aspect of the invention, a membrane extraction method ofdetermining concentration for determining concentration of a componentin a sample is described. The calibrant is added to a stripping fluidprior to contact of said stripping fluid with a membrane. The strippingfluid containing the calibrant is contacting one side of the membraneand sample the other side. During the contact the mass transfer of thecomponent to the stripping phase and calibrant to extraction phaseoccur. Then the stripping phase is introduced into an analyticalinstrument for determination of amount of the calibrant and the compoundpresent in that phase, which is used for calculation of theconcentration of the component in the sample using ratio of masstransfer coefficients corresponding to the calibrant and the component.The membrane extraction and the calibration approach is particularlyuseful for continuous extraction process.

Additionally, according to the invention there is provided a method ofdelivering a compound into investigated system. The method comprisingadding a well defined amounts of said compound into coating of a fiberprior to exposure of the fiber to said investigated system, which couldbe analytical instrument or living animal or a plant. Using fibers fordelivery of the compound reduces risk of exposure to toxic compounds.The compound delivered to instrument can serve as a calibrant andprovide appropriate response factor for that instrument to thatcompound. Alternatively the compound can be a drug or other bioactivecomponent and it can be delivered directly to tissue of living system.

The method of the invention may be used for measuring or identifying oneor more component of interest in an animal or animal tissue comprisesthe steps of: positioning a fibre within the animal or tissue, whereinthe fibre is at least partially coated with an extraction phase foradsorbing the component of interest from the animal or tissue. Theextraction phase is positioned within said animal or tissue, and thusthe component of interest is adsorbed onto the extraction phase for apre-determined period of time. Following this, the fibre is removed fromthe animal or tissue; and the component of interest is desorbed from theextraction phase into an analytical instrument for measurement oridentification.

The method of the invention is suitable for pharmacokinetic studies,wherein observation of analyte levels in a biological system over timeis desirable to conduct with little or no blood or biological fluidremoval from the system. In the case where blood samples would normallybe drawn periodically for pharmacokinetic studies, the inventionadvantageously allows similar observations without removal of bloodvolume from the subject.

The extraction phase may specifically adsorb the one or more componentof interest, and is preferably located at a terminal end (or “distal”end) of the fibre.

The period of time for which the fibre is positioned within the animalor animal tissue can be any acceptable time allowing adsorption of adetectable amount of the component of interest. For example, this timemay be equivalent to equilibration time for a component of interest, orit can be less than equilibration time for a component of interest.

The component of interest can be any desirable component. For example,it may be a bacteria, viruses, sub-cellular components, biopolymers,DNA, proteins, drugs, drug metabolites, hormones, vitamins,environmental contaminants, chemicals, or cells. Any component capableof detection can be selected.

The animal or animal tissue can be is selected from the group consistingof single cell animals, live eggs, mice, rats, rabbits, dogs, sheep,pigs, monkeys and humans. As discussed further herein, an embodiment ofthe invention requires only samples, and is not necessarily conducted inan animal or in animal tissue. The animal tissue could be, for example,isolated cells and organs.

The fibre may be positioned within a blood vessel, and this embodimentwould allow analysis of a component of interest adsorbed from bloodflowing through said blood vessel. Optionally, the step of positioningsaid fibre comprises guiding the fibre into position within the bloodvessel using a catheter. Other areas in an animal in which the fibre maybe positioned include a) muscle, brain, soft tissue, or organ of saidanimal; and the component of interest is adsorbed from interstitialfluid or intracellular fluid; b) an inner part of spine, scull or bone;and the component of interest can be adsorbed from the bone, innerfluids including spinal fluid, bone marrow or brain fluid; or c) a cellof an animal, and an adsorbed component is extracted from the innercellular fluid or sub-cellular component of a single cell of an animal.Of course, the invention is not limited to these examples.

During positioning, the fibre may be disposed within a housing having asealed penetrating end. In this case, the method may include the step ofopening the penetrating end once the fibre is positioned as desiredwithin the animal, exposing the extraction phase within said animal.

Alternatively, the fibre may be inactive during said positioningfollowed by activating the extraction phase using change of electricalpotential or optical means to allow adsorption of said component ofinterest. An example of this could be if the fibre is made of a metalwhich can be activated to attract certain components. Otherpossibilities for electrical activation of the fibre are within thescope of the invention.

The invention may use one fibre, or a plurality of fibres arranged as anarray or bundle. As used herein, discussion of a fibre in the singulardoes not preclude the use of more than one fibre, or a bundle of fibres.In the case where a plurality of fibres are used, they may be disposedin a single position within the animal, or they may be disposed in morethan one position within said animal, so as to obtain readings frommultiple locations simultaneously. The fibre may be one or more opticalfibres, such as a bundle of optical fibres.

In one embodiment of the invention, the extraction phase mayadditionally comprise a strongly bound calibrant which is retained inthe extraction phase during the step of adsorbing. Alternatively, aweakly bound calibrant can be used which is released from the extractionphase during the step of adsorbing according to convection conditionsand diffusion coefficient. The amount of the weakly bound calibrantremaining after the pre-determined period of time can be observed. Thiscan also be used to deliver a desired compound to the animal or animaltissue.

In another embodiment, a strongly bound reagent may be added to theextraction phase prior to extraction. This reagent may be a stronglybound reagent which reacts with the component of interest. An example ofsuch a strongly bound reagent is one that labels the component ofinterest with a fluorescence tag. Another example is a reagent such asan enzyme, in which case the component of interest may be a substratefor that enzyme. Such an enzyme may be one that digests a proteindirectly onto the fibre, for example trypsin or a trypsin cofactor.Further, the reagent may be added to the extraction phase after the stepof adsorbing, in which case the reagent subsequently reacts with thecomponent of interest.

The reagent can be added to the extraction phase by spraying or dippingthe reagent onto the extraction phase.

The method of the invention may be one in which a polymerase chainreaction (PCR) is conducted directly on the extraction phase. In such anembodiment, the components of interest are DNA or DNA fragments, thefibre is subject to periodic cycles of alternating cooling and heating,the reagent comprises polymerase and nucleic acids, and the methodresults in a polymerase chain reaction (PCR) on the extraction phase.

The reagent may comprise an ionization matrix utilized in matrixassisted laser desorption and ionization (MALDI). MALDI analysis of theextraction phase can be conducted with any embodiment of the inventionamenable to such a method of measurement or compound identification. Anynumber of analytical instruments may be used with the invention, such asa spectrometer such as a time of flight instrument mass spectrometer(TOFMS) or an ion mobility spectrometer. After desorbing the componentof interest from the extraction phase, measurement or identification ofthe component may occur in an analytical instrument such as a gaschromatograph, a liquid chromatograph, a capillary electrophoresisinstrument, a capillary electrochromatography instrument and amicrofluidic device.

The invention may include positioning of the fibre in an analyticalinstrument after the step of adsorbing. This could, for example involvelaser irradiation of the fibre to desorb the component of interest fromthe extraction phase into the analytical instrument. In such a case, thefibre can be irradiated in a region not coated with the extractionphase, so as to desorb the component.

The invention may allow introduction of the fibre directly into a massspectrometer prior to the step of desorbing. The fibre may be introducedinto a mass spectrometer by insertion into a small solvent volume in ananospray needle, followed by the step of desorbing, and electrospray ofa desorbed component of interest.

After removing the fibre from the animal or tissue, the fibre may beexposed to a high voltage resulting in field desorption of the componentof interest directly from the extraction phase into the massspectrometer.

Separation of components of interest from the extraction phase may occurdirectly in a separation capillary or channel of the analyticalinstrument. The step of desorbing may be conducted in a small borecartridge filled with a desorption solvent following by automatedmeasurement or identification of a component of interest in theanalytical instrument. In such a case, the fibre may be placed in thesmall bore cartridge immediately following the step of removing thefibre from the animal or tissue, and the cartridge can either beanalysed immediately or sealed and transported or stored prior toautomated measurement or identification.

The invention also relates to a device for adsorbing one or morecomponent of interest from an animal or animal tissue. The devicecomprises one or more fibre having an at least partially coated end. Theend is at least partially coated with an extraction phase for absorbingone or more component of interest. The device also includes apositioning device for guiding the at least partially coated end of thefibre into position within the animal or animal tissue.

Optionally, the fibre diameter can be of millimeter to nanometerdimensions, and formed of any acceptable material that would be amenablefor use in the intended application. Such materials may include fusedsilica, plastic, carbon or metal wire. The fibre may be a plurality ofoptical fibres formed from fused silica.

Optionally, the fibre may be a hollow tubing having the extraction phasecoated on an inside surface of the tubing. In this instance, the tubingmay be in communication with a pump capable of draw up or ejecting asample from the tubing. The pump may be of any acceptable type known foruse with tubing. Alternatively, the fibre may be a hollow tubing havingthe extraction phase coated on an outside surface thereof. In this case,the tubing could be sealed at one end and have a pump in communicationwith the tubing to blow fluid, such as a gas or liquid, into the tubing.This would allow expansion of the tubing as desired, which couldincrease the surface area of the extraction phase as required.

The device of the invention may additionally comprise a sheathsurrounding the fibre for protection and easy handling.

The extraction phase is advantageously biocompatible, as necessary.Optionally, the fibre may be additionally at least partially coated witha biocompatible protection layer, which can surround the extractionphase. Such a biocompatible protection layer may comprise polypyrrole orderivatised cellulose, or any such polymer as would provide protection.

The extraction phase itself may comprises any composition capable ofbinding a component of interest. It may, for example be a polymericcomposition such as substituted or unsubstituted poly(dimethylsiloxane), polyacrylate, poly (ethylene glycol) or polypyrrole.Alternatively, the extraction phase may have a bioaffinity agent on itssurface, such as a selective cavity, a molecular recognition moiety, amolecularly imprinted polymer, or an immobilized antibody. Theextraction phase may contain any of these in combination.

The extraction phase can, alternatively be an extraction and ionizationmatrix for MALDI-TOFMS analysis, and may contain a calibrant molecule,as discussed above.

The fibre may be contained in a housing closed at one end, for openingand exposing the fibre when appropriately positioned within the animalor animal tissue. Such a housing may be a sealed leaf structure, or anyother such openable sealant.

The positioning device itself may a catheter, for those applicationswhere the fibre is guided into a blood vessel, such as a vein, or othertubular biological structures, as discussed in more detail below.Further, the position device may be an x-y-z micro positioning stage,for those applications wherein a tissue can be positioned on such astage, and its movement finely controlled. The positioning devicecomprises an automated system, which may be rendered attachable to theanimal or animal tissue. The positioning device may additionally be usedto position said fibre within an analytical instrument for desorption ofthe component of interest from the extraction phase. The positioningdevice can optionally be used to place the fibre directly inside aseparation capillary or channel, and could be used to couple the fibreto a laser beam facilitating desorption of a component of interest fromthe extraction phase. The positioning device may be used to facilitatedesorption of a component of interest into an analytical instrument.

In the case where a plurality of fibres are used, these fibres may havethe same or a different extraction phase coated thereon, so that morethan one component of interest can be detected. More than one extractionphase can be combined on a fibre, so that a variety of components ofinterest can be detected.

The device may additionally comprise an agitator to cause movement ofthe coated end of a fibre, for example axial or horizontal movement ofthe fibre. In the case where the fibre comprises hollow tubing havingthe extraction phase coated on an inside surface of the tubing, theagitator may force the tubing to draw up a sample into the tubing. Thiscan be effected by mechanical means or by creating a pressuredifferential forcing the tubing to draw up a sample into the tubing. Theagitator may comprise an inflatable balloon.

The invention further relates to a method of measuring or identifyingone or more component of interest in liquid samples arranged in aplurality of wells in a multiwell plate. This involves simultaneouslysubmerging a distal end of a plurality of fibres within the plurality ofwells, respectively, the distal end of each fibre being at leastpartially coated with an extraction phase for adsorbing the component ofinterest from the liquid sample. Following this, the component ofinterest is adsorbed onto the extraction phase for a pre-determinedperiod of time. The fibres are then simultaneously removed from thewells, and are positioned in an analytical instrument for desorption,and measurement or identification of the component of interest from theextraction phase. Such an analytical instrument may be any of the onesnoted above, such as a MALDI analytical instrument or a multichannelmicromachined microfuidic device.

The inventive device for measuring or identifying one or more componentof interest from liquid samples arranged in a plurality of wells in amultiwell plate, for use with the method described herein comprises aplurality of fibres, each having an at least partially coated distalend, said end being at least partially coated with an extraction phasefor absorbing the component of interest. A positioning device is usedfor guiding the coated distal end of said fibres into a submergedposition within the plurality of wells of the multiwell plate, forremoving said fibres from said wells, and for positioning said fibresinto an analytical instrument.

According to one embodiment, a small sterile device containing a smalldiameter fibre with an associated extraction phase coated thereon isused. The extraction phase has affinity for one or more compounds ofinterest. After exposure of the extraction phase in vivo, the device maybe removed for quantitative or qualitative analysis in an analyticalinstrument.

A device for in vivo study of chemical concentrations consists of afibre or wire and associated extraction phase. The fibre or wire may bemade of fused silica, metal, carbon, graphite or a polymeric material.The device may or may not have an attached or removable handle. Thedevice may have an associated or removable housing such as an outerneedle sheath to provide access for the device to the tissue understudy. Preferably the device is introduced to the tissue under study viaa standard medical positioning device such as a catheter ormicrodialysis cannula. After extraction the housing may be retained ifit is used in association with desorption, or discarded if it is notso-needed. Where a medical device is used to provide access to thetissue under study, multiple devices may be used with a single catheterfor instance, obviating the need to puncture the skin or other tissuesseparately for each extraction.

A process of carrying out in vivo solid phase microextraction uses afibre with associated extraction phase, which may or may not have anassociated housing. In any case a means is provided to position thedevice in the tissue for the desired extraction. For extraction thedevice is left in contact with the tissue under study for a sufficientperiod of time to allow equilibration with the chemicals in the tissue,insensitivity to convective forces and/or maximal sensitivity. It islikely the device could be used to monitor chemical concentrations inhumans or experimental animals such as rats, mice, dogs, sheep orrabbits. Subsequent to sampling the device is placed in an appropriateanalytical instrument or desorption device so that at least one chemicalcomponent extracted is desorbed for quantification.

The device and process described are used to monitor chemicalconcentrations in vivo in a living animal, without causing a disruptionin the dynamic balance in the animal systems. Some specific benefits canbe described. Because no blood need be drawn for the analysis, animalsare less stressed. This would allow for more data points to be collectedfor pharmacokinetic profiles, allowing for better data on which to makedrug design decisions. It would also allow or for sampling of blood ortissue drug concentrations at multiple sites in an animal, to betterassess the effects of differing metabolic processes in differentlocations in animals. Where more data points are collected from oneanimal, a reduction in inter-animal variation in the results arises.This variation can often obscure real pharmacokinetic trends and so byeliminating it, better pharmacokinetic data can be collected.Conventional sampling where a specific sample of blood/tissue is removedfrom an animal causes a disruption in the normal chemical balance of theanimal. Each successive sample enhances the impact on the normaldynamics of the animal. With sampling according to the invention, whereonly a negligible portion of the analytes of interest are removed, thenormal chemical balance remains unperturbed, thus eliminating the effectof sampling itself on the results. Genetic variation in drug metabolismwithin a population gives rise to differing pharmacokinetics for thesame drug among individuals. The device and process described would bebeneficial both in monitoring the effect of genetic variation onmetabolism of existing drugs, and for directing the design of noveldrugs to take advantage of variable genetic profiles for tailored drugdesign.

Calibration of the device may be achieved in several ways. Whereequilibrium extraction is achieved, calibration by comparison to matchedin vitro samples is simple and effective. Under non-equilibriumextraction or where it is not possible to match in vitro samples to thein vivo system, calibration may be achieved by pre-loading the fibrewith a suitable calibrant. Direct quantification based on analytephysico chemical properties is also possible using spectroscopicanalysis of the analytes directly from the fibre.

Optionally, calibrant can be added to the extraction phase to performinternal calibration to quantify components of interest. The rate ofloss of calibrant from the extraction phase is used to estimate variousdegree of convection in the system and the matrix affect facilitatingaccurate in-vivo or in vitro calibration.

The device accomplishes both sampling and sample preparation during invivo analyte extraction. Sample preparation may be limited to isolationfrom sample matrix and concentration in the extraction phase. It mayalso include additional processing on the fibre. Examples of this arederivatization of analyte to a form with higher sensitivity in detectionthrough either a modification of product polarity or fluorescenttagging, amplification of analyte copy number in the case of DNAanalysis to improve signal intensity, and protein or enzymatic digestionin the case of general biomolecules (eg. proteins) to convert them to aform more amenable to instrumental analysis (eg. peptide fragments). Inall cases the goal of this on-fibre processing is to enhancedetection/quantification of the target analytes.

In the conventional SPME device the overriding goal in device design wasoptimizing the affinity of the analyte for the extraction phase on thefibre, to maximize analytical sensitivity. In the case of in vivoanalysis the issue of coating biocompatibility is equally important.Device design must take into account both biocompatibility and affinityin the extraction phase.

Because of the simplicity inherent in both the device design and theprocess, multiplexing in both sampling and analysis is much morepractical that it has been for conventional analyses. Fibres may begrouped together in bundles, with fibres having either the same ordifferent coatings, allowing for both sampling and quantification frommany fibres at once, rather than one at a time.

Another advantage of the device and process is that quantification isperformed separately from sampling, using conventional high sensitivityinstrumental analysis. This allows better sensitivity and selectivitythan are achievable where the detection is coupled directly to thesampling/sample preparation as is the case for biosensors. An interfaceis used to couple the fibre to the analytical instrument. This may be assimple as the off-line desorption of analytes into solvent filled wellsin a multi-well plate, to a more sophisticated dedicated interface forthermal, field, solvent or laser desorption. In the case of a dedicatedinterface for solvent desorption, small internal diameter coupled withefficient solvent flow enhance desorption kinetics so that analytes maybe removed from the fibre as quickly as possible.

Although the discussion thus far has focused on using a device withoutcompounds of interest initially loaded into the extraction phase, toinvestigate chemical concentrations in a living system, the devicedescribed is equally suited to the delivery of a precise amount of achemical compound to a precisely targeted tissue. If a device is firstloaded with a pre-determined amount of compound of interest, it can beaccurately positioned at the site of interest, where compounds will moveout of the device according to kinetic and/or thermodynamic principlesand thus supply the chemical to the tissue. This would be of value intargeted drug dosing where only a specific tissue is exposed to a drugcompound.

FIG. 1, part A illustrates an extraction device 1 consists essentiallyof an extraction phase 4 coated on a fibre or wire 2 to be used with apositioning device to accurately locate the device in a tissue. Theentire device is sterilizable by one or more of the conventional meansof sterilization, such as autoclave, ethylene oxide, UV or gammairradiation. The uncoated end of the wire may or may not include ahandle 8 to facilitate positioning of the device. The length of the wireis variable 7 depending on the application requirements. The extractionphase 4 could be a polymeric layer prepared on the wire surface,particulate adsorptive or absorptive material glued or otherwise affixedto the wire surface, or immobilized biorecognition agents such asantibodies nucleotides or protein receptors. When constructed of thestainless steel wire described below the extraction device is quiteflexible. It will follow curves in a vein or catheter and normallyresume a straight configuration when removed. The device is useful forthe application of monitoring concentrations of drugs and theirmetabolites in blood or other tissues, either in single point monitoringor in multiple point (time course) monitoring.

FIG. 1, part B illustrates standard medical catheter is shown inschematic form having a catheter body 10 and a sealing septum 12 (PRN).PRN is the commonly used term for an i.v. adapter to seal a catheter,incorporating a piercable septum, marketed by Beckton Dickinson. In thetext that follows applications are described that use such a catheterfor intra venous (i.v.) sampling. In practice, catheters are availablefor accessing other vessels as well, so applications are not limited toi.v. ones. For instance arteries, vessels within organs or capillariesmay also be accessed using similar devices.

FIG. 1, part C, illustrates an embodiment comprising the extractiondevice alone with no support rod and no handle may be introduced to ablood vessel through a previously placed medical catheter 10 withattached PRN 12. The end of the extraction device with the extractionphase 4 may be contained in a sterile hypodermic needle that is used topierce the PRN and provide access to the catheter. The extraction deviceis pushed partly into the catheter by means of the support wire 2 andthe hypodermic needle is withdrawn. In this case the PRN provides a sealaround the device to prevent blood loss. The extraction device 1 is thenpushed into the catheter and blood vessel by an appropriate amount sothat the extraction phase is exposed to the flowing blood. The catheteris then flushed with saline to prevent clotting in the catheter. Afterthe required time for the extraction of drugs and metabolites thehypodermic needle is once again used to pierce the PRN to provide a portfor removal of the extraction device. The extraction device is thenremoved from the housing, rinsed and packaged for transport foranalysis. The coated fibre can be placed inside a micro-syringe asdescribed in U.S. Pat. No. 5,691,206, for easier handling with acatheter or other positioning device.

FIG. 2 shows the use of a medical catheter 10 passing through the skin20 and vein wall 18 to position the extraction device 9 with PRN 12inside a vein 22 with blood flow 16 past the exposed extraction phase 4.In this position the extraction device has been fully depressed throughcatheter so that the extraction phase is fully exposed to flowing bloodoutside of catheter. PRN 12 is still accessible to allow for flushing toensure patency of the catheter.

FIG. 3 illustrates a schematic of the polypyrrole polymerizationreaction. As an example of an extraction phase, polypyrrole may bedeposited onto the surface of a fine metal wire by electrolyticoxidation under conditions of controlled potential The polymer can beprepared on thin stainless steel wire as described below. The resultingpolymer can then serve as the extraction phase to extractpre-concentrate drug compounds directly from blood flowing in a vessel.An exemplary preparation of coating a stainless steel wire withpolypyrrole is provided in Example 1.

Medical Sampling Device

In use it may be desirable to provide a housing or sheath to allowaccess to the tissue site of interest. The housing is also important toensure correct positioning of the device at a specific location in thetissue or site under study. This may be by puncture of the skin and/orblood vessel followed by positioning of the phase at a specific site foranalysis, incorporation of multiple fibres and agitation means. Thehousing may also be required to provide a seal to prevent blood fromescaping past the device during sampling. The nature of the associatedhousing will be dependent on the site to be sampled.

FIGS. 4 to 9 provide schematic illustration of options for the devicesand described herein.

FIG. 4 shows modifications to the device and a housing for multi-fibresampling using a commercial catheter. Fibres 24, 26 and 28 are coated bycoating 30, 32 and 34 respectively, which can be the same type ofcoating to increase capacity of the device, or preferentially each fibrehaving different highly selective coatings, such as antibodies designedto recognized only defined components of interests in a living animal.

The device may also be used for sampling from an unpressurized medicalport such as a microdialysis cannula. Because such a port is notpressurized, there is no need for a seal to prevent fluid from flushingpast the device during sampling, which obviates the need for anadditional sheath or specialized housing during sampling. The device hassignificant advantages over conventional microdialysis sampling becauseit is not necessary to either add or remove fluid from the tissue tosample. In conventional microdialysis analysis a portion of the fluidthat diffuses into the cannula from the surrounding tissue may beremoved for analysis. Alternatively synthetic fluid is pumped into thecannula and then to an analytical instrument for semi-continuousmonitoring. In both instances the fluid balance of the tissue isdisrupted during sampling, by reduction in volume in the first instanceand by dilution in the second. Analysis using the device according tothe invention would not disrupt the biochemical balance in the tissue asit does not cause such an imbalance.

FIG. 5 shows a modified housing in part A and extraction device in partB appropriate for sampling directly from soft tissue. When the device isin the retracted position the housing as seen in FIG. 5, part A, isconstructed of a rigid tube 40 with a handle 46 and has a sealed tip 44for penetrating soft tissue. The tip is constructed from two or moreleaves separated from each other part way up the housing by a cut orslot 42 and are normally held together by spring action to seal the tip.FIG. 5, part B shows the extraction device supported in a thick tubing 6for opening up the leaves of the needle end to allow exposure ofextraction phase for sampling.

FIG. 6 shows a schematic of the use of the extraction device and housingfor soft tissue sampling. FIG. 6, part A, shows the extraction device 47within the housing 45 in retracted position. FIG. 6, part B, shows theextraction device 47 in the housing 45 in exposed position. Thesupporting wire 6 moves with extraction device to force open the leavesat tip of needle to allow extraction phase on wire to pass through.

FIG. 7 illustrates the housing 45 and extraction device mounted in thex-y-z positioning device 60 consisting of the “z” vertical positioningstage 52 with high resolution dial 54 and the x-y stage 55 withappropriate dials 56 and 58 allowing precise positioning of theextraction phase 4 within the sample 50. This positioning system istypically with microscope to monitor insertion and sampling process. Thehousing is first used to prepare a channel for the device at therequired position for sampling (FIG. 7, part A). The housing is thenwithdrawn slightly while the extraction device 47 is held still. In thisway the extraction phase of the device comes into contact with thetissue surrounding the channel prepared by the housing, thus avoiding aplug of tissue from traveling into the housing, and avoiding having theextraction device itself have to bore the channel in the tissue. In thiscase the device is used to monitor the concentrations of chemicals inthe interstital or intracellular fluids in the tissues, as it would notsample chemical that is bound to tissue proteins or membranes. Thiswould be preferred to tissue biopsy both in terms of the simplifiedsampling and reduced tissue damage.

FIG. 8 shows the catheter with the hollow fiber 38 coated on the insidewall surface at the lower portion 70 of the fiber. The schematic crosssectional view shows the two layer coating 66 and 64 on the inner fibersurface 62. The outer coating 66 is chosen to be biocompatible toeliminate absorption of proteins, while the inner coating 64 is theextraction phase facilitating removal of well defined components fromsample introduced to the inner fiber via channel 68. The sample is drawninto the hollow fiber by using the device 72 generating pressuredifferential, such as syringe or metering pump connected to the hollowfiber. The action of drawing and ejecting sample produces agitation andtherefore accelerate the extraction rate. The tubing is mounted incatheter, but can also be mounted in a positioning device illustrated inFIG. 7.

FIG. 9 shows the catheter with the hollow fibre 38 and stretchablecoating sealing one end that can be blown out forming a small balloonstructure 75 using the pressurized gas delivery device 74, such as smallcompressor or cylinder with carbon dioxide and micro-regulator connectedto the free end of the hollow fibre. The material of the coating or itsmodified surface 76 can be designed to extract compounds from sample.The expended coating has higher surface area resulting in extractionrate enhancement. In addition repeated expansion and retraction of thecoating cause induction of the convection currents and further increasein the extraction rate.

Miniaturization

While the device described is quite small (127 μm diameter), furtherminiaturization would be beneficial, particularly for the study ofsingle cells. As probe size is reduced, the effect of the size of thetheoretical boundary layer around the extraction phase on the rate ofextraction is diminished, as is the case with microelectrodes (Heinze,J. Angew. Chem. Int. Ed. Engl. 1993, 32, 1268-1288.). In practicalterms, this means the degree of convection in the sample has less effecton the rate of extraction. This is important for sampling of any systemwhere static extraction must be conducted, as it would in single cells,or where degree of agitation is variable as it is for intravenoussampling. In addition, the dimension of the extraction phase alsoimpacts extraction equilibration. Thinner extraction phases equilibratefaster and are less dependent on sample convection. Devices with overalldimensions in the range of 1-10 μm would be suitable for monitoring theinterior of single cells while devices in the sub-micron range would beuseful for monitoring organelles within cells. There are currently nofeasible means to accurately assess chemical concentrations occurringwithin cells. All currently available methods either require that thecell is killed (eg. cell lysis followed by CE of cytosolic components inmicrochannels), which may produce an erroneous result, or suffer frompoor accuracy (fluorescence tagging of specific compounds). The mainstrength of the coated fibre technology is that it can monitor cellularprocess in a non-disruptive manner. Only a negligible portion of thechemical is removed, allowing cellular processes to continueunperturbed. Commercially available micropositioning devices using x-y-zstage coupled to microscope can be used to position coated end of thefibre in the well defined part of the investigated system.

Technology that has been developed for genetic manipulation of cellsuses fine capillaries to sample and introduce genetic material in cells,controlled by micromanipulators and monitored by stereomicroscopes.Cells are maintained in isotonic environments during the manipulations,typically by being contained in dishes or vials filled with suitablebuffers. Similar instruments could be employed for positioning andsampling cells with fibre probes.

Portable Automated Sampling

Because the device and process described simplifies sampling and samplepreparation significantly, it provides the opportunity for automatedsampling of tissue concentrations without the need for continual humaninvolvement. In on-line microdialysis sampling an animal being monitoredis tethered to a stationary support and tubing conducting fluid to andfrom the microdialysis cannula and analytical instrument (CE or LC) isincluded in the tether. In the embodiment, an animal being monitoreddoes not need to be tethered, but rather can carry a device forautomatically moving probes in and out of a catheter, cannula or othersampling port at prescribed times. After sampling the device would holdthe probes for retrieval and quantification at a later time. Thisembodiment would have similar advantages to the microdialysis system interms of reduced human intervention and hence reduced sampling errors,with the additional advantage that animals in a study would be lessrestricted and stressed, and experiencing a more normal environment.This would reduce stress impacts on the integrity of the results.

Strategy of Single Use Devices

Up to now SPME devices have been designed to be re-used numerous times.While it is possible to re-use the polypyrrole coated fibre (wire)device described above, it is advantageous that this device be employedas a single-use device. Particularly in implementations where the deviceis exposed to blood, it would not be practical to clean the device andassociated housing sufficiently for re-use. The goal of manufactureshould be to minimize cost so that users find it cost-effective todispose of the device after use.

Coating Strategies

There are a number of additional coating strategies that would bedesirable in the design of these devices, under certain circumstances.These would extend the usefulness of the devices for the purposesdescribed and allow them to be applied for additional purposes.

Improved biocompatibility in the extraction phase would be beneficial toextend either the time period the phase can be in contact with tissues,or increase the number of samplings that can be made from one site. Thiscan be achieved in two different ways. Either new phase with betterbiocompatibility could be selected or a biocompatible outer layer couldbe used in conjunction with an inner extraction phase having lowerbiocompatibility.

Polypyrrole itself has good biocompatibility. It has been used forseveral years in biosensor devices without any evidence of toxicity,immunogenesis (initiation of an immune response) or thrombogenesis(initiation of clotting response). It is an example of an extractionphase that is suitable for exposing directly to the investigated system.If it is desirable to use a less biocompatible extraction phase thedevice could be rendered biocompatible by coating the extraction phasewith an outer biocompatible layer such as derivatized cellulose.Analytes of interest would diffuse freely through this outer layer andbe extracted by the extraction phase on the inner layer. This may beuseful if more traditional extraction phases such as poly(dimethylsiloxane), polyacrylate or poly (ethylene glycol) are ofinterest for extractions.

Biorecognition entities that either comprise the extraction phase or areimmobilized in another phase having low extraction affinity couldprovide both higher selectivity and higher sensitivity in theseanalyses. Higher affinity would provide higher sensitivity and moreeasily allow for shorter probe residence times. Higher selectivity wouldallow for reduced disturbance of the system under study, furtherenhancement of sensitivity and reduced concern for competition inextraction. This would permit the quantitative analysis of one compoundpresent at low concentration when a competing compound is present athigh concentration.

Biorecognition in the extraction phase may be accomplished by entrapmentof antibodies or another molecules capable of biorecognition in an inertbiocompatible extraction phase. This is demonstrated this in the use ofpolypyrrole to entrap antibodies specific for diazepam.

FIG. 10 shows a chromatogram comparing extraction of a sample containingdiazepam, with a device with polypyrrole only, versus a device withentrapped anti-diazepam antibody. In this case the analyte affinity tothe antibody is much higher than it is to the polypyrrole. Alternativelyantibodies, nucleic acids or other molecules may be covalently attachedto the fibre using typical immobilization strategies or they may beelectrostatically immobilized by means similar to the immobilization ofnucleic acid to nitrocellulose used in current blotting technologies.For covalent immobilization either random or oriented strategies may beused in one application or another.

FIG. 11 shows a schematic of the oriented immobilization of antibody 172on a surface 170, and attraction of antigen 174, to form anantibody-antigen complex 176. The diazepam may be liberated from thecomplex for quantification by temporary or permanent denaturation of theantibody protein.

If a probe with very high selectivity was developed, it couldpotentially extract only the compound of interest, which would eliminatethe need for chromatography in the analysis. Direct introduction to amass spectrometer for quantification would further simplify theanalytical process. Such entities may include antibodies or antibodyfragments, proteins, protein subunits or peptide sequences, DNA, RNA orpolynucleotides or the antigens or substrates that bind with any ofthese. Such biorecognition entities may be immobilized by adsorption,electrostatically, covalently or by entrapment within another matrix.Covalent immobilization may be by either random or oriented means.

Biorecognition may also be achieved by using molecular imprintedpolymers. In this case a polymer is prepared in the presence of theanalyte of interest. The polymer contains functional groups thatinteract electrostatically with the analyte. After polymerization theanalyte is removed and cavities remain in the polymer, with appropriatefunctional groups located inside. When used for extraction, analytefreely soluble in the sample is attracted to the cavities and held thereby electrostatic forces. These polymers are seen by some as syntheticantibodies due to their high selectivity for the analyte of interest.Such polymers provide enhance selectivity when used as extraction phasesin devices according to the invention.

It is also possible to prepare a coating that can have its extractionefficiency gated or activated just prior to extraction. This would allowfor the pre-positioning of the device in a specific site, and thenactivate the extraction phase just as extraction is to start. Becausepolypyrrole is a conducting polymer, this may be accomplished byapplying a small charge to the fibre. This is useful for the extractionof ionic compounds through controlling of the oxidation state of thepolymer. Alternatively this may be accomplished using the device shownin FIG. 8 for soft tissue sampling. The device could first be positionedin the desired location, but the exposure of the fibre to the tissuecould be delayed until the proper time to initiate sampling.

Use of Indicator Compound

A common and valuable tool in bioanalytical analysis is the monitoringof the appearance or disappearance of an indicator compound that isspecific for a biochemical pathway. This is used for instance to monitorfor the presence of specific cells or bacteria, or for the presence offree enzymes. In the typical chemical reaction a substrate (S) istransformed into a product (P) by interacting with a single enzyme or anenzyme system with associated cofactors. Enzymes may or may not betransformed in this process. The indicator may be the substrate, inwhich case its disappearance is monitored, or it may be the product, inwhich case its appearance is monitored. The amount of indicator formedin a specific time is correlated to both the amount and activity of thetarget enzyme present. If the indicator has an affinity for theextraction phase, enzyme activities and/or metabolic rates may bemonitored in situ. The substrate may be either loaded onto the fibre orplaced into a cell suspension or enzyme solution. When the fibre isplaced into the solution, indicator will become immobilized in thefibre, and can be subsequently quantified by an analytical instrument.

Pre-Loading of Fibre with Calibrant

For conventional SPME analysis, a common difficulty is in devisingaccurate means of quantification. For in vitro analysis quantificationis often achieved by adding a known amount of standard to the sample,and then performing the analysis. This is referred to as calibration byinternal standard or standard addition. The amount of the standardrecovered is assumed to be correlated with the amount of unknown analyterecovered and the ratio is calculated in order to determine the originalconcentration of unknown. For in vivo and in situ analysis it istypically not practical to add a standard to the system under analysis.Until now the most practical means of calibration is by preparing aseries of synthetic standards that match the sample as closely aspossible, and comparing the results from the standards analysis withthat of the unknown. This approach was described above for thecalibration of polypyrrole devices in the in vivo pharmacokinetic studywith reference to FIG. 12. In this case whole dog blood was obtainedfrom a commercial supplier and samples were prepared with various drugconcentrations. Upon analysis a calibration curve is constructed andthis curve is used to interpolate unknown detector responses to estimateunknown drug concentrations. While the method is conceptually simple, itis not always highly accurate as it cannot accommodate the impact ofslight changes in the in vivo site for impact on the results.

As an alternative to conventional internal standard calibration, astandard may be loaded onto the fibre (extraction phase) prior toanalysis and the loss of standard from the fibre is monitoredinstrumentally. Where the kinetics of absorption of the internalstandard analyte to the fibre is equivalent to the kinetics ofdesorption (binding is reversible), absorption and desorption arecontrolled by diffusion in the sample and the rate of loss of standardfrom the fibre will be correlated with uptake of analyte by the fibre.The amount of analyte lost may be correlated with the amount absorbed,and consequently with sample concentration of unknown also. Using thisstrategy variation in sample convection may be controlled for byreferencing unknown analyte to the amount of calibrant lost from thefibre. Alternatively, where the convection conditions and hence rate ofmass transfer and are known or controlled, the use of an irreversiblybound calibrant on the fibre may be used. The fibre would first beexposed to a matrix-matched standard with a known concentration ofanalyte. The fibre would subsequently be exposed to the unknown sample.The ratio of unknown to standard extracted by the fibre would accuratelyreflect the ratio of unknown to standard sample concentrations. (G.Xiong, Y. Chen and J. Pawliszyn “On-site calibration method based onstepwise solid-phase microextraction”, J. Chromatogr. A 999, 43-50(2003)).

Pre-loading of compound onto the fibre may also be used for calibrateddelivery of compound to a precise tissue region. Where the compoundpre-loaded has low to moderate affinity for the fibre, compound willpartition out of the fibre and into the surrounding tissue duringexposure. This may be used as a means of dosing only one targeted tissueregion with a drug or other compound of interest, avoiding dosing of thewhole animal as is commonly the case in therapeutic drug regimens.Tissue dosage control may be attained by precisely controlling theexposure time. Dosage may then be confirmed by desorbing remaininganalytes into an analytical instrument to quantify the amount remaining,allowing the calculation of the amount delivered.

An exemplary use of calibrant is discussed below with reference toExample 4.

Standards in the Extraction Phase

Internal standard and standard addition are important calibrationapproaches, which are very effective even when quantifying targetanalytes in complex matrices. They compensate for additional capacity oractivity of such a matrix. However such approaches require delivery ofthe standard to the matrix. This adds additional steps to the samplepreparation, which makes the process longer and is sometimesprohibitive, for example in the case of on-site or in-vivodeterminations.

According to an aspect of the invention, an alternative approach may beused where the standard is delivered together with the introduction ofthe extraction phase. This approach is not practical to implement forexhaustive extraction techniques, since large volumes of the extractionphase having a high affinity for the target analytes are used in theseapproaches to facilitate as complete removal of the analytes from thematrix as possible. However, in microextraction a substantial portion ofthe analytes is present in the matrix during the extraction and afterequilibrium is reached. This presents an opportunity to add the standardto the investigated system together with the extraction phase. Forexample, when performing small volume (a few mL) sample analysisinvolving microextraction step, as is frequently the case with automatedanalysis, placing the extraction phase/standard mixture in the vial withthe sample can be combined with addition of a standard. In this way, thestep of spiking the sample with a standard is eliminated.

Desorption and re-equilibration of a standard originally present in thematrix can occur simultaneously with the mass transfer and equilibrationof the target analytes from the matrix to the extraction phase. Thus,the standard delivery process according to this aspect of the inventiondoes not add substantially to the extraction time. For example, inautomated fiber SPME analysis the standard can be introduced onto thecoating during an automated analysis process by exposing the fiber to avial containing the standard. Alternatively, multiple fibers containingstandards can be used each for single analysis. In addition, thestandard can be generated in or released from the coating by way of achemical reaction in the coating.

A significant impact of the standard in the extraction phase approachfor calibration would be realized for on-site, in-situ or in-vivoinvestigations. However, it is desirable to minimize the amount offoreign substances added to the investigated system. For this reason,direct standard spike into the matrix is typically not possible. Fullre-equilibration of standards present on the fiber is frequently notfeasible because of the potential for contamination and large dilutionwhich could occur in on-site investigations. However, successfulcalibration can be accomplished by investigating kinetics of thedesorbtion/sorption process. Since the rate of extraction for mostpractical types of extractions is controlled by mass transfer throughthe boundary layer, the desorbtion rate or the standard can be used asan indication of the extent of the boundary layer (existing either inthe matrix or in the extraction phase, or both). This information can beused for calibration of the target analytes.

In an advanced approach, standards can be added to balance the analyteloss from the matrix during extraction, similar to methods which may beused in dialysis, in order to minimize the impact of standards on theinvestigated system. This objective may be accomplished by adding thesame amount of the standard as the amount of analyte being removed fromthe matrix. The standard chosen may be an isotopically labeled analog ofthe target analyte, in order to minimize impact on the investigatedsystem. In addition, this approach can allow study of thephysicochemical partitioning and adsorption phenomenon among samplematrix components.

When employing a standard in the extraction phase, calibration can beaccomplished in any microextraction or steady state approach includingSolid Phase Microextraction (SPME), micro liquid phase extraction(MLPE), and membrane extraction or headspace extraction. In SPME, astandard can be used to dope the solid/polymeric extraction phase. InMLPE, a standard would be present in the liquid extraction phase. Inmembrane extraction, the standard would be present in the striping phaseand in headspace extraction in the gaseous headspace. In some cases thestandard can be delivered by other components of the extraction system.For example, in the fiber SPME the standard can be delivered from aneedle by first sorbing the standard onto the needle material. Also thestandard could be delivered together with the vial, for example byincluding the standard on or in the wall of the vial.

An exemplary use of calibrant is discussed below with reference toExamples 6, 7, 8, 9, 10 and 11.

Use of Multiple Fibres

The development of multiple fibre coating strategies has severalbenefits. In addition to providing more flexibility in selecting devicesfor a particular application, multiple devices could be used in parallelto provide for a more complete profiling of the types and amounts ofcompounds present in a sample. This may be accomplished either byexposing multiple fibres to a sample in parallel, or by preparing onefibre with multiple sorbents.

Fibres for Conducting Micro-Chemical Reactions

On-fibre reaction can significantly enhance the detection of componentsof interests. For example on-fibre fluorescence labeling has been hasimproved detection limits for detection of toxins at trace level (A.Namera, A. So, J. Pawliszyn “Analysis of Anatoxin-a in Aqueous Samplesby Solid-Phase Microextraction Coupled to High-Performance LiquidChromatography with Fluorescence Detection and On-Fiber” DerivatizationJ. Chromatogr. 963, 295-302 (2002)). Two of the most important chemicalreactions for molecular characterization in genomics and proteomicsresearch are DNA amplification and enzymatic protein digestion. Bothprocesses are enzymatic reactions that are conducted in vitro, with theproducts either being carried on to a further processing step oranalysed directly.

In DNA amplification a small number of DNA or polynucleotide fragmentsare amplified by the enzyme DNA polymerase. Through the action of theenzyme and suitable substrates, the copy number of DNA fragments can beincreased exponentially in just a few hours. The process ischaracterized by high fidelity so that the end product is a very puresolution of identical DNA fragments. Typically the amount of DNAoriginally present is insufficient for further processing and/oranalytical characterization whereas the concentration in the finalproduct is sufficient. The product may either be characterized fornucleotide content and sequence or used for the preparation of peptidesor proteins coded by the DNA sequence.

For enzymatic protein digestion a protein sample is digested by enzymesthat cleave the polypeptide chains at specific sites. The resultingpolypeptide fragments may be characterized for molecular weight orpeptide content and sequence. Typically the intact protein is too largefor direct characterization and so a protein is characterized by a‘fingerprint’ analysis of the pattern of polypeptide fragments producedby one or more enzymatic cleavages. Alternatively the polypeptides maybe sequenced and the sequence of the original protein reconstructed.This allows for example, that the DNA sequence coding for the proteinmay be determined either for the purpose of identifying its location inthe genome or for development of an expression system to produce theprotein in quantity.

With the continued miniaturization of genomic and proteomic analysesthrough the use of micromachined or μTAS devices, there is a need tominiaturize the sample preparation and introduction steps that come upfront. These types of miniaturized analyses are increasingly importantin the fields of genomics and proteomics where sample sizes are smalldue to the high cost of these samples. Also the miniaturization allowsfor parallelization and higher throughput in analysis to moreefficiently process the very large number of samples made possible bythe completion of the human genome project. A porous polymer attached toa fine fibre or wire makes an ideal medium in which to conduct theseenzymatic reactions in miniature scale, with the added advantage thatwhen the reaction is complete, the device is also suitable forintroduction of the reaction products directly to a microanalyticalsystem.

Interfaces

As described above one of the strengths of the device and processdescribed is that once sampling and sample preparation(pre-concentration and elimination of matrix) have been completed, thedevice of the instant invention is ideally suited for directlyintroducing the extracted analytes to an instrument for separation andquantification.

Conventional SPME devices are interfacing to GC or LC equipment forquantification of amount of compound extracted. In the case of GCequipment the fibre is exposed in the heated injection sleeve similarlyto the way a conventional syringe injection is conducted. Analytes forGC analysis are necessarily volatile at the temperatures normally usedin a GC injector and are efficiently desorbed in the hot carrier gasflowing through the injection sleeve and into the separation column.Compounds analysed by LC are typically non volatile and/or thermallyunstable and so heat cannot be used for desorption. For LC desorption adedicated interface is required to first remove analytes from the fibreand transfer them to a solvent. A portion or all of this solvent is theninjected into the instrument for analysis. In the commercial interfacethe fibre is desorbed in a solvent filled chamber in a valve connectedto the instrument inlet. After desorption the valve is switched in linewith the pressurized solvent flow of the instrument and the entirevolume of the desorption solution with dissolved analytes is introducedto the instrument.

Modification for Efficient LC Quantification

The technique has been limited by the relatively large volume of thecommercial desorption interface (100 μL). Because of the phase thicknessof the commercial SPME devices for LC (ca. 50 μL) this large volume isrequired. If desorption volume is reduced a significant proportion ofthe analytes are not removed from the fibre. Carryovers in the range of20% are common (depending on the specific analyte and desorption solventused) as the volume of desorption solvent is reduced below 50 μL. Thesevolumes, however, are too large for typical LC applications as injectionvolumes are in the 10-20 μL range, particularly for LC/MS applications.Large injection volumes in these analyses typically produce unacceptablybroad chromatographic peaks and poor resolution. When only a smallportion of the total desorption solvent is injected, inferiorsensitivity results. One strength of device of the instant invention isthe ability to introduce all of the extracted analyte to the instrumentfor quantification. This allows for maximal sensitivity. Fibres withsignificantly reduced phase thicknesses, such as the polypyrrole coatedwire described for the pharmacokinetic analyses, may be efficientlydesorbed in 10-20 μL of desorption solvent. The entire desorption volumemay then be injected for quantification. The result is sharp,symmetrical peaks as are shown in FIG. 13, which may be accuratelyintegrated and produce good chromatographic resolution.

The foregoing described the use of static desorption, but dynamicdesorption of analytes is also of interest in certain applications. Thisis achieved by passing desorption solvent over the fibre duringdesorption. Because the fibre is continuously exposed to freshdesorption solvent, quantitative desorption is theoretically possible.The rate of desorption is governed by the rate of solvent flow over thefibre. Faster flow results in faster desorption. To achieve the fastestdesorption possible and to avoid ending up with an overly large solventinjection plug, it is necessary that the inner diameter of thedesorption chamber is as small as possible. When volumetric flow isconstant, faster linear flow is achieved in a smaller diameter chamber.This results in a shorter desorption time and hence a minimized totaldesorption volume.

Automation of LC Quantification

While the reduced volume HPLC interface used to date allows forefficient transfer of analytes from the fibre to the instrument, theprocess is only partially automated. To date the introduction andremoval of the probe wire to/from the interface must be performedmanually for each injection.

FIG. 14 illustrates a micro-cartridge 77, which contains coated piece offibre in its small cavity 79 and sealed with plugs 78. The cavity 79 canbe filled with desorption solvent. After extraction the coated piece offibre containing the coating 4 is placed in a cavity 79 of the cartridge77 for protection during storage and transport. Determination ofextracted components can be performed in an automated instrument adoptedfor use with cartridges.

Interface for CE, Use of Electrokinetic Stacking

As discussed above, the device of the instant invention provides anideal means for interfacing sampling and sample preparation tomicroanalytical instruments, particularly when devices much smaller thanthe commercially available SPME devices are employed. In capillaryelectrophoresis and related technologies, analytes are separated in acapillary typically 50 μm in diameter. This is too small forconventional syringe injection. Injection is typically by hydrodynamicor electrokinetic means. With hydrodynamic injection a sample is placedin the buffer reservoir associated with one end of the capillary. Thatend is then lifted above the opposite end by a prescribed amount for aprescribed time. The volume of sample entering the capillary may becalculated from the time, the elevation difference, the capillarydiameter and the solution viscosity. The sample solution is thenexchanged for running buffer solution prior to applying the separationvoltage. While simple, the technique suffers from inaccuracies ininjection volume and poor reproducibility from one analysis to the next.With electrokinetic injection a sample is again placed in the bufferreservoir associated with one end of the capillary. An injection voltageis applied across the reservoir and capillary and analytes in solutionmove into the capillary by electromotive force. Once sufficient materialhas been injected the voltage is removed and the sample solution isagain exchanged for running buffer solution prior to applying theseparation voltage. This method suffers from inaccuracy in injectionsdue to the variation in electrophoretic mobility between analytes. Thisresults in different amounts of the different compounds present beinginjected. A small diameter fibre with extracted analytes may beintroduced directly inside a CE separation capillary filled with runningbuffer (Whang, C. W., Pawliszyn, J. Anal. Commun., 1998, 35, 353-356).This allows for accurate, quantitative introduction of analytes forseparation.

As an improvement to this technique for CE analysis, by carefullymatching the outer diameter of the fibre and the inner diameter of theseparation capillary, a stacking of analytes occurs prior to separationresults. This allows for much superior resolution. Electrophoreticvelocity is inversely proportional to the cross-sectional area insidethe separation capillary. When this area is reduced, increased velocityresults because of an increase in electric field gradient. When a fibreis introduced inside a CE capillary, the space between the fibre and thecapillary wall has a much smaller cross-sectional area than the spaceafter the fibre where only buffer is present in the capillary. When afibre is present and separation voltage applied, the analytes move outof the fibre and along the restricted channel quite quickly. When theyreach the area of open capillary mobility drops significantly and theanalytes are concentrated in a narrow band. During separation a higherresolution is achieved than would otherwise be possible.

FIG. 15 illustrates x-y-z positioning device for use with a fibrebundle. Individual fibres may be positioned precisely in the separationcapillary prior to desorption. In this case the extraction phase wouldbe coated on more than just the very tip of the fibre, as is shown inFIG. 15 (132) and desorption would be accomplished by applying anappropriate electric potential rather than by laser pulsing.

Direct Introduction to MS through Nanospray Nebulizer

In some instances it is not necessary to chromatographically separateextracted analytes prior to quantification. This is the case where thefibre has very high selectivity such that only the analyte of interestis extracted with no interfering substances. It is also true where massspectrometry is used for detection/quantification and components areseparated by mass rather than by time prior to quantification. For MSapplications it is possible to place the fibre directly into a nebulizerneedle in an electrospray ionization source.

FIG. 16 describes this process schematically. Solvent flowing throughthe nebulizer 150 efficiently desorbs analytes from the fibre 164 priorto being nebulized and sprayed in a plume 156 in a mass spectrometeratmospheric pressure ionization source 160. Ionization is thenaccomplished by standard ESI with MS detection, ie. droplets in theplume 156 are dried and reduced in size by hot gas flow 152 until ions154 form in the vicinity of the orifice 166. These then pass into themass analyzer 162 in the instrument.

Application to MALDI Analysis

Matrix-assisted laser desorption/ionization (MALDI) is a technique forionization of molecules using a laser as the energy source. As a verysoft ionization method, MALDI yields primarily the singly chargedprotonated molecule which are then conveniently quantified by either ionmobility spectrometry or time of flight mass spectrometry. This featurehas made MALDI a widespread ionization tool for high molecular weight,nonvolatile and thermally labile analytes. MALDI has enabled the routinedetermination of large bimolecular such as peptides and proteins (P E.Jackson, P F. Scholl, and J D. Groopman, Molecular Medicine Today, 2000,6, 271.)

The embodiment of the invention wherein the inventive fibre device iscoupled to MALDI advantageously allows a combination of sampleextraction with the ionization procedure on the very tip of a fusedsilica optical fibre for bimolecular analysis. The sample end of thefibre was coated for the extraction of peptides and/or proteins in amatrix solution. In the case of enkephalin and substance P the matrixused was alpha-cyano-4-hydroxy cinnaminic acid. The optical fibre thusserved as the sample extraction surface, the support for the sample plusmatrix, and the optical pipe to transfer the laser energy from the laserto the sample. Laser energy was transferred through the other end of theoptic fibre to ionize and desorb the biomolecules for subsequentanalysis. This fibre/MALDI combination was coupled with an ion mobilityspectrometer and a tandem quadrupole/time-of-flight mass spectrometer(in separate experiments) for the detection of the MALDI signal.

FIG. 17 shows a schematic of the fibre/MALDI-IMS interface andinstrument. This consists of a laser source 96 and focusing lens 80,which focuses the laser light onto the uncoated end of the fibre, heldin an x-y-z positioning array 84. The array movement may be manual orautomated. The fibre 86 transmits light from the source to the x-y-zpositionable inlet 88 of the mass analyser 90, which in this case was anion mobility spectrometer. In the inlet 88 the coated end of the fibre86 is held in place by two silicone septa 94 and a section of supporttubing 92. Only the very tip 98 of the fibre is coated with extractionphase. A photosensitive diode 82 is positioned at the laser source 84 tosense the desorption laser pulse and initiate data collection 100.

FIG. 18 shows the ion mobility mass spectrum of enkephalin and substanceP were obtained using this system.

One advantage of the MALDI/IMS interface is that the MALDI source isoperated at ambient pressure instead of high vacuum, as it is inconventional MALDI/TOF mass spectrometery. Also, loss of sampledelivered to the drift tube is negligible at ambient 115 pressure and ithas been reported recently that atmospheric pressure MALDI produces agenerally uniform ion cloud at atmospheric pressure. The ionizationprocess is even softer than that of the conventional high vacuum MALDIand is capable of producing protonated molecular ions for smallproteins. This is convenient for the MALDI analysis of macromoleculesbecause of the relative absence of metastable fragmentation anddiscrimination in the ionization process compared to conventional vacuumMALDI. The most promising advantage of this ambient interface is thepossibility of interchangeably using the same instrument for bothelectrospray and MALDI sample introduction.

FIG. 19 shows a schematic of the laser desorption interface an ionformation. In this case two time of flight mass spectrometers (TOF) areused, one to sample positive ions 110 and the other to sample negativeions 112. A laser pulse initiates desorption from the extraction phase114 and polarized plates 116 accelerate the appropriate ions into theappropriate mass analyzer.

Though MALDI has enabled the routine determination of large bimoleculessuch as peptides and proteins, it has always been great interest todevelop quantitative MALDI analysis. For quantitative work withconventional MALDI analysis, the laser beam is scanned cross the samplearea on the target plate, and each sample spot is irradiated withmultiple laser shots until a striking decrease in signal detection isobserved which indicates the removal of most of the sample loaded onthis particular spot. Therefore, tens to hundreds of laser shots must befired to finish the scanning process, and the final spectrum istypically a sum or an average of all the spectra obtained from eachlaser shot. This sampling process will lead to the unavoidable poorshot-to-shot and spot-to-spot sample reproducibility, and has beenconsidered as the fundamental limitation for method quantification inMALDI analysis.

The combination of the inventive device with MALDI has technicallysolved the above problem as it combines sample extraction with theionization procedure on the tip of a fused silica optical fibre. Theoptical fibre thus served as the sample extraction surface, the supportfor the sample plus matrix, and the optical pipe to transfer the laserenergy from the laser to the sample. Since the sample was loadeddirectly on the fibre tip, so the sample size was identical to that ofthe laser irradiance area and there existed no spot-to-spot desorptiondifference. In addition, due to the multiple reflections inside thefibre, the primary laser profile is converted into a homogeneousintensity profile at the sample end fibre surface. This means that laseremission is homogeneous through the fibre tip surface. The method wasdeveloped as to accomplish all sample desorption that was extracted onfibre tip with a single laser shot. As long as this situation could besatisfied, the spot-to-spot and shot-to-shot spectral disparity wouldalso be minimized. In this way it dramatically improved thequantification aspect of MALDI as well as saved large amount ofanalytical time and analyte consumed. To explore the quantitativeaspects of the fibre/MALDI method TOAB was selected as the analytecompound and all experiments were performed in the matrix DHB. Thefibre/MALDI-IMS system described in FIG. 17 was used for quantification.

In the extraction step of the previous experiments, the tip of thefibre/MALDI fibre was dipped into the solution containing both sampleand matrix. For this pre-mixed extraction method, the analyte to matrixratio was pre-optimized and fixed for the best performance and this isalmost impossible for the detection of analyte in real samples ofunknown concentrations. Meanwhile, due to the very small capacity of theextraction phase, there exists a competition between the analyte andmatrix that causes a further limitation for the amount of analyte thatcan be extracted. A more practical way is to load the matrix in a secondstep after sample extraction. Spray method with a nebulizer is an idealcandidate for this purpose as it forms very fine solution drops smallerthan 100 nm. After sample extraction, matrix solution is loaded with anebulizer. The fog like matrix drops would help to form more uniformcocrystalline on the fibre tip surface. The amount of matrix loaded onthe fibre or matrix to analyte ratio could be easily adjusted by varyingthe concentration of the matrix solution and the spray time.

Example 2 describes use of a MALDI/IMS interface which is associatedwith reduced noise. Reduced noise, though convenient, is not necessary.Thus, careful alignment of the laser with the sample surface isoptional, as the fibre itself can accomplish this. As an alternativehowever, it would still be feasible to conduct a conventional MALDIanalysis where the laser is directed at the surface of the fibre. Thiswould allow devices to be constructed from non-light conducting fibres,and would eliminate the need to optically couple the device and thelaser prior to analysis.

Multiplexing for Parallel Extraction and Quantification

The inventive device described lends itself to parallelization in boththe sampling and quantification steps, due to both its cylindricalgeometry and simplification of the analytical process.

FIG. 15 illustrates that parallel sampling could be accomplished bybundling multiple fibres, with the same or different coatings, to eitherprobe multiple samples at once or to probe a single sample for multipleanalytes. The bundle of fibres could also be used to provide efficientstirring during extraction. The extraction can be from multi-wellautosampler plate, each well containing a different sample is extractedby a single fibre facilitating highly parallel determinations. Thebundled extraction device could be employed for quantification by theMALDI process described above. The bundle could be multiplexed to alight source, and each individual probe irradiated in sequence bytargeting the source at each individual fibre in succession.Simultaneously the sample end of each fibre would be positioned at theinstrument for analysis. As shown in FIG. 15, a laser source 120 isirradiated in sequence onto each fibre in a fibre bundle 130 by means ofa positioning device 122. The sample ends of the fibres in the bundleare directed into an extraction/desorption mesh 126. In this case onlythe tips of the fibres are coated with extraction phase 132 as this isthe surface that is irradiated by the laser light. The sample end ispositionable by means of a second positioning device 124. As each fibreis ready to be desorbed, it is positioned by the positioning device 124at the sampling orifice of the mass analyzer 128 and the laser 120 isfired to intimate desorption.

Alternatively the probe bundle could be desorbed simultaneously intoindividual solvent desorption wells, with quantification by LC/MS.

The combination of fiber MALDI analysis with multiwell plates may alsoinvolve a positioning device to allow proper placement of the distal endof each coated fibre within a small opening of each well, so as tosubmerge the extraction phase. This approach requires design of arelatively small and accurate positioning device, to accommodate thelarge number of wells in a single high density multiwell plate. Thetechnology now allows for over 1,000 wells to reside on one plate. Otherintroduction techniques may be used to introduce a sample or fibre intoa well, specifically by using micromachined microfluidic systems wheremany microfluidic channels can be placed in one microfluid device toaccommodate each fibre. This can be performed in combination withnanospray introduction to MS, where all fibres are desorbed in parallelin a microstructure, and subsequently each desorbed solution isintroduced to MS in sequence.

EXAMPLE 1 Preparation of Polypyrrole Coating on Stainless Steel Wiresand Use in a Biological System

Stainless steel wires (grade T-304V, 0.005″) were from Small Parts Inc.(Miami Lakes Fla.). Lithium perchlorate (95%) and pyrrole (98%) werefrom Sigma/Aldrich (Mississauga, ON). Pyrrole was used as received forone month after opening, was stored refrigerated and the bottle waslayered with nitrogen after each use. Polypyrrole (PPY) films weredeposited onto the supporting electrode surface (stainless steel wire)by anodic oxidation of the pyrrole monomer in the presence of an aqueouselectrolyte solution (counter ion). A potentiostat/galvanostat (Model273, EG&G Princeton Applied Research) was used for theelectrodeposition. The last 15 mm of the wires were coatedpotentiostatically at 0.8 V for 20 minutes. The placement of a siliconseptum 15 mm from the end of the wire allowed for accurate control ofcoating length. The coating solution used was pyrrole (0.1M) and lithiumperchlorate (0.1M) in water and was prepared fresh daily. Coating wasperformed in a custom designed 50 mL flow-through glass compartment.Coating solution was pumped through the compartment continuously toallow for one complete change of solution during each deposition (50mL/20 min.). The stainless steel wires were cut into 10.7 cm sectionswith a razor blade and 2-4 cm at the end to be coated was etched with400 grit silicon carbide polishing paper. Wires were then sonicated inacetone until required to prevent accumulation of oxides or othercontaminants on the wire surface. Immediately before use the wires wererinsed briefly with water and were installed as the working electrode.The counter electrode consisted of a ca. 10 cm section of platinum wire(0.75 mm OD) formed into a coil of about 1.5 cm diameter. The stainlesssteel wire was placed into the coating solution in the centre of thiscoil. A calomel reference electrode was used. The polypyrrole coatingthickness was estimated to be <10 μm thick. Prepared probes were thenplaced into vials with sufficient buffer to cover the extraction phaseand autoclaved for sterilization.

Wires prepared as described above were characterized in a series of invitro experiments. Benzodiazepine standards (1 mg/mL in methanol) werepurchased from Cerilliant (Austin Tex.). These were diluted in methanolto prepare mixtures of various concentrations for use in samplepreparation and instrument calibration. Samples were prepared frombuffer, dog plasma or dog blood and spiked with an appropriate amount ofthe analytes of interest. The device was placed directly into the samplecontained in an appropriate polypropylene sample vial, for a certainperiod of time. After extraction the probe was rinsed briefly with astream of water and either analysed immediately or allowed to dry priorto analysis. Drugs were stable in the extraction phase when stored dryat room temperature for at least 24 hours.

FIG. 20 and FIG. 21 show two alternatives for device response that maybe achieved by this method. In FIG. 20 it can be seen that a fastinitial equilibrium between the sample and native polypyrrole coatedwires. After a longer period of time additional analyte is extracted asthe polymer swells and exposes additional sites for extraction.

FIG. 21 shows that the polymer was preconditioned with methanol toprovide a swelled polymer prior to extraction. The result theelimination of the initial lag time seen in FIG. 20 and an immediateincrease in amount extracted with maximal extraction seen after 30minutes when the analyte has diffused throughout the bulk of the polymerto access the additional sites exposed during swelling. This providesfor additional sensitivity at the expense of a slower response time.

FIG. 22 shows the result of in vitro extraction calibrations from bufferand plasma. Because the device will only extract unbound drug andbecause the drugs under study are ca. 90% bound to protein, the plasmaconcentrations tested were 10× higher than the buffer concentrations. Inbuffer 100% of drug is free and 0% is bound to protein as no protein ispresent. In plasma, it is expected that 10% or less of the drug will befree.

FIG. 22 demonstrates that the linear range attained is similar in bufferand plasma, based on free drug concentration. The figure alsodemonstrates that polymer extraction reaches maximal capacity in asolution with 100-200 ng/mL free drug.

After extraction (either in vivo or in vitro) the compounds on thedevice are desorbed in a small volume (10-20 μL) of desorption solvent,75% methanol in this case. Maximal desorption is seen in as little as 20sec. All or a portion of the desorption solvent is injected to ananalytical instrument for analysis. This may be accomplished eitheron-line in a dedicated injection interface that takes the place of theregular injection port on a LC, or off-line in a small desorptionchamber, followed by standard syringe injection of the desorptionsolvent by a commercial autosampler.

FIG. 12 presents the results of a calibration from whole blood treatedwith an anticoagulant. The figure demonstrates good linearity inextraction over the range of total (bound plus free) drug shown.

FIG. 13 shows a chromatogram obtained after extraction of drugs spikedat 100 ng/mL from dog plasma, demonstrating the good chromatographicpeak shape obtainable by the method. In this case the injection volumewas ca. 11 μL, using the desorption solvent described above.

FIGS. 23 to 26 show the results of the use of the device by the cathetersampling method described above, for a pharmacokinetic study in dogs. Inthis case dogs were dosed with diazepam at time 0:00. Multiple samplingswere performed from a catheter over the ensuing 12 hours. Calibrationwas by comparison to results from an external calibration in whole bloodsimilar to that shown in FIG. 12. Also shown is a comparison to resultsobtained by multiple blood draws over the same time period, withconventional sample preparation and analysis as described in thedescription of the prior art. These results demonstrate that the deviceis useful for the application described and that the method describedproduces results in good agreement with devices and methods usinginvasive prior art sampling techniques.

Table 1 shows the limits of detection achieved in buffer and whole bloodfor a “probe” formed according to the invention. As can be seen fromthese data, the device and method allow an extremely sensitive detectionof the analytes of interest, in this case: diazepam, nordiazepam andoxazepam.

TABLE 1 Limits of Detection Achieved in Buffer and Whole Blood detectionlimit linear Compound Linear Range (S/N = 3) ng/mL slope correlation(r²) SPME probe calibration from whole blood Diazepam 1–1000 ng/mL 7.1215 0.999 Nordiazepam 1–1000 ng/mL 3.1 328 0.994 Oxazepam 1–1000 ng/mL2.7 258 0.996 SPME probe calibration from buffer Diazepam 10–100 ng/mL0.43 306 0.999 Nordiazepam 10–100 ng/mL 0.24 281 0.998 Oxazepam 10–100ng/mL 0.35 169 0.995

EXAMPLE 2 MALDI Analysis

In this Example, a medical aerosol compressor was used as the matrixsprayer, and 10 mg/mL matrix DHB solution was deposited into thenebulizer vial. After analyte extraction the fibre tip was placed 1.5 cmabove the nebulizer vial, and by turning on the compressor very finedrops of the matrix DHB solution were formed and attached to the fibretip. The 800 μm fibre was tested with the spray method for a 0.05 mg/mLTOAB sample solution. The times for matrix application were set at 45seconds and 30 seconds, respectively, considering the lower analyteconcentration. Two 3 minute air-dry times were applied before and afterthe spray of matrix.

FIG. 27 shows an IMS spectrum from this analysis. The limit of detectionwas found to be 0.02 mg/mL with S/N ˜2. This level is 10 times lowerthan the previous 0.2 mg/mL established by the 400 μm fibre using thepre-mixed method. The sensitivity has been increased dramatically. Thisgreat improvement was attributed to the larger surface area as well asthe spray method.

In the work described above the laser pulse was shot down the core ofthe fibre. In addition to the advantages associated with reduced noiseas described above, this is convenient as it is not necessary tocarefully align the laser with the sample surface. The fibre itselfaccomplishes this. As an alternative however, it would still be feasibleto conduct more of a conventional MALDI analysis where the laser isdirected at the surface of the fibre. This would allow probes to beconstructed from non-light conducting fibres, and would eliminate theneed to optically couple the probe and the laser prior to analysis.

EXAMPLE 3 On-Fiber and in-Needle Laboratory

In micromachined devices, controlling flow is not simple since itrequires pumps or electroosmontic flow means. In addition it is quitedifficult to mix analytes in the small channels. A more efficientapproach is to do sample processing on the surface or in thin layersadjacent to the surface. The structures chosen in this Example are theouter surfaces of fibers. Alternatively, the inside surface of a tubefiber could be used. This Example makes use of a small fiber todemonstrate a convenient sampling method to collect analytes from smallobjects. In this work capillary electrophoresis with fluorescencedetection has been used to facilitate detection of small amounts ofanalytes extracted by the fiber.

Chemicals and materials. 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)was purchased from Fluka (Sigma-Aldrich Canada Ltd., Oakville, Ontario).Brij35® and all amino acids (glycine, L-phenylalanine, L-proline,L-glutamate and L-aspartate) were obtained from Sigma-Aldrich CanadaLtd. (Oakville, Ontario, Canada). Sodium borate was from FisherScientific (Nepean, Ontario, Canada). All of the solvents used were HPLCgrade, filtered and degassed and all the aqueous samples were preparedwith deionized water (NANOpure, Ultrapure water system). A manual SPMEassembly and replaceable extraction fibers, coated withCarbowax-temprated resin (CW-TPR, 50 um) were purchased from Supelco(Canada).

Instrument. The high voltage power supply for the CE system was fromSpellman High Voltage Electronics Cooperation, Plainview, N.Y., USA. TheCE separation capillary and silica fibers were purchased from PolymicroTechnologies LLC, Phoenix, Ariz., USA.

The fundamental components of the laser induced fluorescence detection(LIF) are the laser, focusing lens, objective lens, interference filterand photomultiplier tube. An Argon ion (Ar+) laser (˜5 mW) was theexcitation source. It provided an excitation wavelength of 488 nm (itsmaximum). The microscope objective lens (10×) and the low pass filter(530 nm) as an interference filter were from Melles Griot (Toronto, ON,Canada). The photomultiplier tube (PMT) and its socket including a highvoltage power supply were purchased from Hamamatsu (R928 and C6271,Bridgewater, N.J., USA). In the design, the optical chopper and lock-inamplifier were used to enhance the signal indirectly. The opticalchopper (SR-540) and lock-in amplifier (SR-510) were from StanfordResearch systems (Sunnyvale, Calif., USA). The analog output signal fromthe lock-in amplifier was read by a plug-in data acquisition card (Star4.5, Varian), which recorded at 20 Hz followed by the digitalization ofthe analog signal.

CE system set-up. The CE system was composed of a high voltage powersupply and a separation capillary with an effective length of 45 cm (75um I.D. and 385 um O.D.). The running buffer was 20 mM sodium borate, 10mM Brij 35® and 2.5% methanol. The capillary was conditioned with 0.1 Msodium hydroxide (NaOH), water and running buffer for 15 minutes each.Between runs, the capillary was reconditioned with 0.1 M NaOH for 4minutes followed by running buffer for 2 minutes. The running voltagewas 12 kV. The injection was done hydrodynamically by raising thecapillary inlet by 5 cm for 5 seconds.

In-solution derivatization reaction. The reaction solution was preparedby mixing 10 ul of each amino acid (0.01 mM in water), 10 ul of 1 mg/mlof NBD-F and 60 ul of 10 mM borate buffer at pH 8. This mixture wasvortexed for 30 seconds and held in a 60° C. water bath for variousperiods of time (2, 5, 10, 30 and 60 minutes). The reaction solution wasdiluted to a final volume of 800 ul with running buffer and stored in anice bath while waiting to be analyzed.

On-fiber derivatization reaction. A fiber was first cleaned by soakingit in ethanol. It was then dipped into a vial containing NBD-F (2-3mg/ml in ethanol) for 10 min with magnetic stirring at 1000 rpm. Afterthat it was transferred to a 1-ml Teflon centrifuge tube containing 200ul of amino acids (0.1 mM) in sodium borate buffer (50 mM, pH 6.0) anddipped in the sample for 20 sec. A 4-ml amber vial containing 1 ml oftriethylamine (TEA) was maintained in a 60° C. water bath. The headspaceof this vial was basic. When the fiber with extracted analytes wasexposed to it, the derivatization reaction took place (15 min).

Whole grape sampling with fiber/CE: interface and off-column desorption.The fiber/CE interface with off-column desorption was describedpreviously. The desorption solvent (2 ul) was placed on the surface ofthe SPME fiber coating. This small droplet was manually rolled aroundthe surface of the fiber coating. Finally this droplet was placed on topof a section of quartz tubing and it slipped down to the other end ofthe tubing where the capillary inlet was fixed. The capillary inletcontacted with this droplet for approximately 2 s. Since this quartztubing was positioned 10 cm above the buffer vials, the droplet washydrodynamically injected. The capillary electrophoresis wassubsequently started. With such an interface, a commercial carbowax/TPRSPME fiber from Supelco could be used.

Fiber/CE interface: on-column desorption. On-column desorption has beendescribed previously for a fiber/CE interface. The SPME micro fiberswere made by attaching a 2-cm long silica fiber (100 um diameter) to a10-cm polyimide coated silica capillary (100 um ID×365 um OD) with epoxyglue. The unit was air-dried overnight. The micro fibers were furtheretched to approximately 50 um diameter with 50% HF. These fibers werefinally housed in stainless steel tubes and were sent to RestekCorporation, Bellefonte, Pa. for coating with carbowax.

Results and Discussion: Separation of amino acid derivatives. Thecritical micellar concentration (CMC) of Brij 35® is 0.9 mM. The CErunning buffer used in these experiments had Brij 35® of 10 mM. Brij 35®not only forms micelles to improve the separation resolution, but alsoenhances the fluorescence intensity of the amino acid derivatives. Somestudies have shown that Brij 35® will enhance the florescence signal ofsuch derivatives by at least three times. The methanol (2.5%) in therunning buffer functioned as organic modifier. It helped to increase thesolubility of the solutes and enlarged the migration time window. As aresult, a better resolution was achieved. Under these conditions, amixture of five amino acid derivatives (phenylalanine, proline, glycine,glutamate and aspartic acid) was analyzed within 20 minutes.

On-fiber derivatization reaction of amino acids. The on-fiberderivatization of amino acids with NBD-F was first established withfibre/HPLC/fluorescence detector system described. On-fiberderivatization and a CE/LIF detection system has been described in theexperimental section. The separation of amino acids derivatives wasestablished. NBD-F reacts with to amines and nucleophilies under a mildbasic condition. The peaks observed of sp-a represented the sideproducts of the reaction of NBD-F and TEA. The peaks of sp-b observedare representative of the side products of the reaction of NBD-F withthe aqueous buffer components and the sample matrix. NBD-OH is the majorside product formed in the reaction of NBD-F with aqueous solution.Glycine could not be analyzed under these conditions because the glycinederivative co-eluted with one of the side-product peaks, sp-b. Glycinehas an average migration time of 6.47 min (RSD=1.37%). Using a similarprocedure, the fibre/CE/LIF detection system was used for the amino acidanalysis. In this study, the fibre/CE with off column desorption wasused.

Whole grape sampling:Off-column desorption fibre/CE interface. Todemonstrate the application of this technology to the direct analysis ofsmall living objects with on fiber derivatization coupled to a CE/LIFdetection system, whole grapes were used as the samples. With a NBD-Fdoped fiber, the sampling procedure and derivatization reaction took 20seconds and 20 minutes, respectively. The resulting electropherogramsfrom this method with green grape (G) and red seedless grape (R)illustrate a glutamic derivative found at 7.05 min for the green grapesample and 7.01 min for the red seedless grape sample. These migrationtimes corresponded to the L-glutamate standard 7.04 min. In the fibre/CEexperiments, most of the peaks were saturated such that phenylalanineand proline derivatives were hidden in the saturated signal. For furtheridentification of amino acids in the sample, the juice from the grapeswas analyzed. The glutamate was also found in the grape juice sample inthe form of glutamic acid.

On-column desorption fibre/CE interface. With the off column desorption,fibers with carbowax/TPR coating were used for sampling, while theon-column desorption was used with microfiber having thinner coating.These microfibers had a diameter between 75-50 um, and so could be usedto sample smaller living objects. These microfibers were coated withcarbowax. Electron microscopy was used to visualize the fibers and thefiber coating was found to be about 10 nm.

The feasibility of using these fibers for the on-fiber labeling reactioncoupled to CE/LIF detection system was tested. First, the blank NBD-Fsolution was extracted with the fiber and desorbed on-column. The dopingof NBD-F was successful. After the sampling of amino acid standard andreaction in the TEA headspace, no product was detected.

EXAMPLE 4 Instrument and Method Calibration Using Fibers Loaded withCalibration Compound

One of the main advantages of the invention is that it allows veryconvenient introduction of extracted components onto analyticalinstrumentation, such as gas chromatography, liquid chromatography,supercritical fluid chromatography, capillary electrophoresis,micro-channel devices and even directly to mass spectrometry anddetection instruments. This feature can be further explored fordelivering calibration standards to analytical instrumentation.Currently standards are delivered to the instrument by injecting thesolvent mixture containing appropriate calibration compounds. However,presence of solvent frequently interferes with calibration procedure.Therefore, it would be to user benefit to eliminate solvents from thecalibration procedure. It can be accomplished by desorbing standardloaded fiber into appropriate instrument.

The loading of the fiber can be accomplished by exposing sorbent-coatedfiber (extraction phase coated) to the source of the standard. Thestandard is then adsorbed onto the fiber coating. Another approach is toimmobilize chemical standards via chemical reaction onto the fiber,which then is released to the instrument under conditions of increasedtemperature, light, chemical potential, mobile phase, etc. The secondapproach ensures stability of the calibrant, but as this Exampledemonstrates the first approach can also be very effective.

Two calibration methods were used. The first approach used solid sorbentcoated fibres. One of the methods is to deliver the calibration compoundby utilizing SPME fibre to the analytical instrument. The standard isfirst extracted from the standard mixture using strong sorbent followedby introduction of the standards loaded fibre into analytical systemrequiring calibration. In this approach liquid injection is avoided andthus solvent interference to the determination of trace VOC (volatilecomponents) is eliminated. Satisfactory calibration curves were obtainedfor the very volatile compounds namely methanol, acetone,dichloromethane and chloroform when a 75-umcarboxen™/polydimethylsiloxane (CX/PDMS) fiber/coating was used. Thestandard gases or gas mixtures of VOCs were prepared using the NISTtraceable certified permeation tubes combined with gas chambers or bymicrowave-assisted evaporation. “Stepwise” is the approach to the secondcalibration method developed during this work for on-site calibration offibres. In this approach the CX/PDMS coated fibre was loaded withstandard followed by exposure to the investigated system and thenintroduction into GC system for analysis. The accumulation time ofanalytes can be controlled equal to or different from that of thestandard, and the response factors for the analytes can be adjustedaccordingly. A good reproducibility of the response factors for BTEX wasobtained with the stepwise method. Satisfactory results were obtained byusing this method in quantitative analysis of BTEX in the gas stationair. The introduction of standard via the stepwise procedure makes thetechnique more useful in field applications. This approach in somerespects resembles standard addition, but also external calibration. Itcan be used to detect leaks, contaminations and losses from the time ofstandard loading onto the fibre to introduction to analyticalinstrument.

Preparation of standard gases or gas mixtures. Up to now several methodshave been developed for preparation of standard gas. Two methods wereemployed in this work to prepare the required standard gases or gasmixtures.

Preparation of gas mixture of BTEX using NIST permeation tubes. Thestandard gas mixture of BTEX (benzene, toluene, ethylbenzene, p-xyleneand o-xylene) was generated with the NIST traceable certified permeationtubes (Kin-Tech Laboratories, La Marque, Tex.) and the gas chambersbuild in our laboratory. It was a flow-through system with which aconstant concentration of standard gas (or gas mixture) can be gained.The temperature was controlled at 50° C. and the air flow rate was at300 ml/min. The gas mixture was sampled from the gas chamber.

Microwave-assisted generation of gas standards of VOCs. A domesticmicrowave oven (1000 W, Model MW5490W, Samsung, Korea) and 1-L gassampling bulbs (Supelco, Bellefonte, Pa.) were used for preparation ofstandard gases and gas mixtures of the investigated VOCs with differentconcentrations. The inner walls of glass bulbs were deactivated bysilanization and the bulbs were cleaned with flushing nitrogen beforeuse. For preparation of standard gases or gas mixtures of BTEX,1,3-dichlorobenzene, 1,1,2-trichloroethane and tetrachloroethylene, aclean piece of glass wool (ca. 10 mg) was set inside the sampling portof the bulb each time and was moistened with deionized water (15 μL).Water was used to absorb microwave energy and then to prompt theevaporation of the compounds that are poor absorbers of microwave. Forpreparation of standard gas mixtures of acetone, methanol,dichloromethane and chloroform, no glass wool and water were needed. Theport of the glass bulb was sealed with a Teflon-faced silicon rubberseptum through which a certain volume of liquid of target compound (ormixture of several compounds) was injected onto the glass wool. Finally,the bulb was placed into the microwave oven to receive microwaveradiation for 90 s. The microwave output was always set to the maximumpower level. After cooling the Supelco bulb to room temperature,analysis of the standard gas was performed through the sampling port ofthe bulb where a septum is located.

The device and the “stepwise” procedure. Fiber coatings and conventionalsamplers used were provided by Supelco (Bellefonte, Pa.). The coatingsutilized included 75-μm Carboxen™/Polydimethylsiloxane (CX/PDMS), 85-μmPolyacrylate (PA), 100-μm Polydimethylsiloxane (PDMS) and 65-μmpolydimethylsiloxane/divinylbenzene (PDMS/DVB).

The stepwise procedure was conducted as follows: first, the fiber wasexposed to tetrachloroethylene standard gas in the bulb, then the fiberwas withdrawn into the needle after 2-min extraction and a ThermogreenSeptum (LB-2, Supelco) was used to seal the tip of the needle. Whenusing the field SPME sampler, no separate sealing septum is needed. Thetetrachloroethylene loaded fiber was then exposed to BTEX standard gasmixture in the chamber or to real air sample for a few minutes. Finally,the fiber was transferred to the GC injector to desorb the standard andanalytes at the same time.

GC-FID analysis of analytes. A Varian model 3500 GC equipped with aflame ionization detector (FID) was employed for sample analysis. ASPB-5 capillary column (30 m×0.25 mm×1 μm) from Supelco (Bellefonte,Pa.) was used and hydrogen as carrier gas at 30 psi. The column wasprogrammed as follows: 35° C. initial, held for 1 min, ramp to 135° C.at 10° C./min and held for 1 min. The detector was maintained at 280° C.For the PA, PDMS and PDMS/DVB fibers, the injector was controlled at250° C. and desorption time was 1 min, while CX/PDMS fiber was desorbedfor 2 min at 300° C.

Comparison of introduction of VOCs standards into GC system by syringeinjection of standard solution and by standards-loaded fiber. Severalvery volatile compounds, namely acetone, chloroform, dichloromethane andmethanol, were selected for investigation. A standard solution wasprepared using methanol as the solvent and the concentration of acetone,dichloromethane and chloroform was 10 μg/ml for each compound. TheGC-FID chromatogram obtained by injecting 0.1 μl of the standardsolution into the GC system illustrated that the solvent peak was toolarge to be well separated from peaks of other compounds and thus madeit difficult to accurately determine those trace components.

On a contrary, there was no big solvent peak appearing in thechromatograms obtained by injecting a standards-loaded fiber into the GCsystem and an ideal separation and identification of the VOCs weretherefore achieved. The analysis of the standard gas mixture wasconducted for 3 min using a 75-μm CX/PDMS fiber and the concentration ofacetone, chloroform, dichloromethane and methanol in the standard gasmixture was 50.5 μg/L for each compound. Due to the avoidance of solventinjection, it became easy to get satisfactory chromatograms for themicro amount of VOC standards, even for the very volatile compounds thatpossess quite short retention times.

In addition, it is difficult to obtain a calibration curve by directlyinjecting pure liquid of individual VOC or liquid mixture of VOCs intoGC system to avoid introduction of plenty of solvent due to thedifficulty of accurate injection of a very small volume (<<0.1 μl) ofliquid standards into the GC system to match the quantitative ranges oftrace analysis.

Calibration curves obtained with this technique for GC analysis of someVOCs. Two different fibers were used to extract the standard gasmixtures of two different groups of VOCs. The very volatile compounds,including acetone, chloroform, dichloromethane and methanol, wereextracted with a 75-μm CX/PDMS fiber, which has a high affinity towardsto VOCs as described above. BTEX were extracted with a 100-μm PDMSfiber. It is known that the PDMS fiber extract target compounds byabsorption while CX/PDMS fiber works by adsorption. By introduction ofthe VOCs standards into GC system with fibers, satisfactory calibrationcurves regarding the concentration-response relationship for SPME-GC-FIDanalysis of the mentioned VOCs were obtained and shown in FIG. 2. Therelated calibration equations were listed below:Methanol: A=767.69 C+3564, R ²=0.9937  (1-a)Acetone: A=3234.8 C+22693, R ²=0.9952  (1-b)Dichloromethane: A=1004.6 C+7271.5, R ²=0.9965  (1-c)Chloroform: A=980.46 C+719.5, R ²=0.9993  (1-d)Benzene: A=106.18 C−1069.5, R ²=0.995  (2-a)Toluene: A32 326.42 C−4320.8, R ²=0.9993  (2-b)Ethylbenzene: A=711.53 C−6136.8, R ²=0.9958  (2-c)p-Xylene: A=868.43 C−10704, R ²=0.9994  (2-d)o-Xylene: A=995.98 C−9588.1, R ²=0.9972  (2-e)where A is the chromatographic peak area (counts) and C is theconcentration of VOCs standard gas (μg/L).

The experimental results demonstrated that the investigated fibers areefficient for introducing VOCs standards into GC system without solventinjection for getting calibration curves and fibre-GC is a highlyfeasible method for quantitative analysis of VOCs, even for the veryvolatile compounds.

Moreover, it is also possible to establish the “mass:response”calibration curves for GC analysis of VOCs by introducing standards withSPME fibers. When absorption-type fibres are employed to extractanalytes, there is a direct relationship between the initial analyteconcentration in the sample (C₀) and the amount of the analyte extractedby the fibre at equilibrium (n) according to Equation 3-a:

$\begin{matrix}{n = \frac{K_{fs}V_{s}V_{f}C_{0}}{V_{s} + {K_{fs}V_{f}}}} & \left( {3\text{-}a} \right)\end{matrix}$where K_(fs) is the fibre/sample partition coefficient, V_(f) is thefibre coating volume and V_(s)the sample volume. For adsorption-typeSPME process, the amount of analyte A extracted by the fibre atequilibrium (n) also grows with the increase of the initial analyteconcentration in the sample (C_(0A)) before saturated adsorptionreached:

$\begin{matrix}{n = {C_{fA}^{\infty} = \frac{K_{A}C_{0A}V_{s}{V_{f}\left( {C_{f\;\max} - C_{fA}^{\infty}} \right)}}{V_{s} + {K_{A}{V_{f}\left( {C_{f\;\max} - C_{fA}^{\infty}} \right)}}}}} & \left( {3\text{-}b} \right)\end{matrix}$where K_(A) is the adsorption equilibrium constant of analyte A, C_(fA)is the concentration of analyte A on the fibre at steady state, C_(fmax)is the concentration of active sites on the surface (corresponding tothe maximum achievable analyte concentration on the surface), V_(s) andV_(f) are the volumes of the sample and the fibre coating, respectively.

Stability of VOCs on extraction phase coatings after exposing thecoatings to zero air. Analytes present in sample by either absorption oradsorption, collect or enrich the target compounds from samples onto thecoatings. However, similar to other extraction procedures, enrichment isfollowed by an opposite procedure, the release of extracted compoundsfrom the coating phase. Therefore, when a coating loaded with some VOCsis exposed to pure air, a part of extracts will transfer to the air andthen the extracts tend to reach an equilibrium distribution between thecoating and air phases. The release of the extracts from the coatingphase depends on a lot of factors, mainly the compounds' and coatings'properties, the temperature of the environment and the differences ofthe compounds' concentrations in the coating phase and in the sample orenvironment. It was shown in Table 2 that the remains of several VOCs(BTEX were included) on the 85-μm PA, 100-μm PDMS and 65-μm PDMS/DVBcoatings ranged from 0 to 91.5% after exposing the coatings to zero airfor 1 min at room temperature. However, the 75-μm CX/PDMS coating couldstore the extracts as much as 89.9%-97.2% even through a 6-min exposureto zero air under the same conditions. Actually, no obvious losses couldbe found for most of the extracts on the 75-μm CX/PDMS coating whenexposure time was controlled within 4 min. Thus it is possible to allowa “stepwise” procedure conducted with the CX/PDMS coating—that is, thiscoating can be used to extract a compound in first step and thentransferred to extract other compounds while the compound extractedpreviously still remains on the coating.

Selection of internal standard for BTEX analysis with stepwiseProcedure. 1,3-dichlorobenzene, 1,1,2-trichloroethane andtetrachloroethylene were tested, respectively, as internal standards forBTEX analysis when a 75-μm CX/PDMS coating was used. The CX/PDMS coatinghas a strong affinity towards these compounds and their storage on theCX/PDMS coating was close to that of BTEX. Considering theirchromatographic behaviors, tetrachloroethylene is the best internalstandard for BTEX analysis since it can be well separated from BTEX andits peak is located in the central position of the chromatogram. Furtherinvestigation demonstrated that, under the selected conditions, theloading of tetrachloroethylene on the fiber did not affect the BTEX, andin turn, the BTEX did not affect the storage of tetrachloroethylene onthe fiber either. Tetrachloroethylene as the internal standard for BTEXanalysis has another advantage—that is, its background is generally notpresent in main BTEX sources like petroleum. However,tetrachloroethylene also has its drawbacks: it is a halogenated compoundand the GC-FID response for it is not as sensitive as for BTEX. Theproblem involved in determination sensitivity for tetrachloroethylenecan be solved by using selective detectors like MSD or FPD.

Response factors for BTEX when tetrachloroethylene was used as internalstandard. For chromatographic analysis, the response factor (F) can bedefined as the following form:

$\begin{matrix}{{\frac{Ax}{Cx} = {F\frac{As}{Cs}}},} & (4)\end{matrix}$where Ax and As are the peak areas of analyte X and internal standard,while Cx and Cs the concentrations of analyte X and standard after theyhave been mixed together. For the use of tetrachloroethylene as internalstandard for BTEX analysis following the stepwise procedure describedabove, the standard is not mixed with analytes before they wereextracted onto the SPME fiber. In such a case Cs stands fortetrachloroethylene's concentration of the standard gas and Cx isindividual BTEX's concentration in the sample.

For the stepwise GC/FID analysis of BTEX, the response factors weremeasured. The time for of BTEX was 2 min, equal to that for thestandard. It can be seen that the response factors highly coincided forduplicate tests in almost all cases. This reflects that the stepwiseprocedure is a feasible and practicable method to introduce internalstandard for GC analysis of BTEX when the CX/PDMS coating is used.

Effect of extraction time on response factors for stepwise procedure. Itshould be noticed that the response factors discussed above are not onlyrelated to the sensitivity of GC-FID determination to individual BTEXbut also depend on the SPME efficiency for them. Since the standard andBTEX were not extracted at the same time during the stepwise procedure,the time for BTEX can be controlled equal to or different from that forthe standard. Obviously, time control will significantly affect theresponse factors. This is one of special features of the stepwiseprocedure distinguishing with the conventional way to use internalstandards. In the conventional way, the extraction of analytes andinternal standard is conducted simultaneously. It was found that theresponse factors varied linearly with the time for SPME of BTEX in therange of 1-5 min when SPME time for tetrachloroethylene (standard) wasconstantly controlled as 2 min. The linear equations obtained were asfollows:Benzene: F=4.265t+1.301, R ²=0.9979;  (5-a)Toluene: F=5.776t+0.402, R ²=0.9981;  (5-b)Ethylbenzene: F=4.663t−0.031, R ²=0.9996;  (5-c)p-Xylene: F=4.623t−0.247, R ²=0.9993;  (5-d)o-Xylene: F=4.767t−0.703, R ²=0.9963,  (5-e)where F is response factor and t is time in minute for BTEX.

The concentrations of both standard and analytes were within the linearranges when the internal standard was used. GC-FID is known to have alinear response to VOCs during a very wide range and so is the inventiveprocedure with the CX/PDMS coating. The excellent linearity of theresponse factors for BTEX varying with the extraction time alsoreflected that the compounds' concentrations studied were located withinthe linear range.

Field application—analysis of BTEX in the air of a gas station. The VOCs(BTEX were the interests) were sampled from a gas station that is 5-minwalk to our laboratory, using the home-made field sampler with a 75-μmCX/PDMS coating. It was a clear day and the temperature was ca. 24° C.when the sampling was conducted. The glass bulb holding the standard gasof tetrachloroethylene (8.1 μg/L) was carried to the field and analysisof tetrachloroethylene was performed prior to BTEX. The sampling timewas 2 minutes for the standard and 4 minutes for the gas station air. Assoon as the sampling was finished, the sampler was delivered to thelaboratory and then the fiber was immediately introduced to GC-FID. Theidentification of individual BTEX was based on their retention times aswell as GC-MS analysis using a Hewlett-Packard 6890 GC equipped with a5973 MSD (Agilent Tech., USA). Tetrachloroethylene was not found in thegas station air itself. The separation of standard and BTEX from othercomponents of the extracts was very good, only one peak might contain m-and p-xylenes, which couldn't be separated from each other under theselected chromatographic conditions. Finally, using the peak areasobtained (As and Ax), the standard concentration (Cs) and the responsefactors (F) given by Equations 5-a-5-e, the concentrations of BTEX inthe air were calculated according to Equation 4.

Conclusion. For getting calibration curves in GC analysis of VOCs inair, fiber was successfully used to introduce VOCs standards into GCsystem without solvent injection. The avoidance of solvent injectionwith the inventive technique made it easy to obtain satisfactorychromatograms for micro amount of VOC standards, even for the veryvolatile compounds that possess quite short retention times. Moreover, astepwise method was developed to introduce internal standard for GCanalysis of BTEX in field application. The CX/PDMS was proved to be theonly suitable coating to fit this method due to its extraordinaryaffinity towards VOCs. Tetrachloroethylene was selected as the internalstandard for reasons such as its proper retention time compared withthose of BTEX for GC analysis, similar behaviors to BTEX on the CX/PDMScoating and very low background in main BTEX contamination sources inthe environments. Using the developed method, analytical results can becalibrated without a necessity to spike standard material into samples,hence it makes the inventive procedure even more advantageous in fieldapplications. However, since the standard is not directly added into thesample and the analysis of standard and analytes is conducted stepwise,this method may not meet the need of calibration of the air matrix'seffects on the analysis of BTEX. This approach is also suitable todetected problems with fiber storage in filed devices, such as leaks,which will result in analyte and standard losses. Further development ofthe technology can include chemical immobilization of compounds, whichwill facilitate production of certified standards.

EXAMPLE 5 Electrophoresis in Non-Uniform Channel Modulated by Insert

Electrophoretic behavior of analyte in capillary consisted of two partswith different cross-section was investigated. The modulation of theseparation path was achieved by inserting into the capillary acylindrical fiber at different depth. The sample loaded at the end oflower cross-section and the appropriate zone, at it was demonstrated,spatially narrowed in the wide capillary part according to the electricfield strength ratio in the two parts of the capillary. Additionally,the low conductive sample buffer can enhance the further signalnarrowness and increase the total probe amount, introduced into thecapillary by electroinjection. The applications of this concentrationtechnique includes focusing after desorption from SPME fibers into theelectrophoretic separation channel prior separation or prior to directdetection using for example UV-Visible, Fluorescence, electrochemical,NMR or mass spectrometry detection. Also, focusing of analytes presentin a buffer is possible by inserting different shapes inserts prior toseparation or direct detection. Periodic insertion of the insert intothe channel will modulate the concentration of analyte, which facilitateseparation and monitoring of the system connected to the separationchannel. The modulation input could be random and the signal can be thenanalyzed by multiplex data processing techniques, such ascross-correlation. Modulation of the diameter of the channel can be alsoaccomplished by applying external pressure or electrical pulses, whichwill also result in focusing without need for movement of the insert inand out of the channel. The results described below indicate that thefocusing occur in a cross channel configuration since the channelcross-section diameter increasing substantially in the area where twochannels meet. This can be used effectively to facilitate concentrationof analyte prior to separation in the second channel, or intwo-dimensional separation when the separation in the first channel isfollowed by focusing at the interface between the two channels prior tosecond dimension separation in the second channel.

Introduction. Non-constant form of a separation channel inelectrophoresis is a way of providing the variance of some parameters(electric field strength, temperature, pH) which can play an importantrole for the process concerned. Smooth form changing is required shouldone need to obtain the appropriate smooth function. By varying across-section of electrophoretic camera one obtains an electric fieldgradient, this gradient can be used both in combination with some otherforce applied, what is used in so-called “gradient focusing techniques”or by itself provided the current density drop and the chamber designare appropriate to form sufficient temperature difference. The lattereffect was used in IEF in thermogradients caused by internal Jouleheating.

A separation channel, composed of few different parts but each one ofconstant form, can be used rather for sample introducing, detecting,multi step analysis development in microarray etc. The results describedin this paper are also important for the methodologies where the sampleapplication procedure is connected with a long object inserting (e.g.microfiber) into the separation capillary.

Experimental: Apparatus. The whole-column imaging detection (WCID) of UVabsorbance was conducted in the iCE280 CIEF instrument (ConvergentBioscience Ltd., Toronto, Canada) with a fixed wavelength of 280 nm. Ashort fused-silica capillary (5.5 cm long) with an ID. of 100 um,internally coated with fluorocarbon (J&W Scientific, Folsom, Calif.),was assembled in a cartridge format (Convergent Bioscience Ltd.) Theentire process of capillary conditioning, sample injection, datacollection, and processing was implemented by a PC computer, and theelectropherogram was recorded as absorbance versus the distance to theanode.

Materials and Chemicals. Optical fiber with a 50 and 61.5 um core(FHP050055065 & FVP60072082) was purchased from Polymicro TechnologiesInc. (Phoenix, Ariz.). pI-markers and buffer chemicals were obtainedfrom Bio-Rad. Water was purified using an ultrapure water system(Barnstead/Thermolyne, Dubuque, Iowa) and was used for all solutions.

Procedures. The fiber was inserted into the capillary at differentdistances and the capillary was filled with running buffer (Phosphate5-100 mM, BioRad). Then, the sample was injected electokinetically, withthe injection time being specially selected to achieve completereplenish of the first the first capillary part (containing an insertedmicrofiber). After, the electrode reservoirs were washed and the desiredbuffer was placed and the electrophoretic run performed.

Results and Discussion. The initial zone width is an important matter inCZE. For the case the sample concentration is insufficient to providesensitive detection, a number of on-line preconcentation procedures isdeveloped. The simplest electrophoresis-based techniques are connectedwith a special conductivity profile creation allowing us to achieve ahigher electric field strength value in the sample zone place, althoughconcentration mechanism may be different (e.g., CE- or ITF-based). Thesimilar effect of electric field enhancing can be obtained due tostepwise cross-section change.

In these experiments, by inserting the cylindrical microfiber thecross-section of the separation channel was modulated. Sample wasinjected electrokinetically at 500v, the duration of voltage pulse wascontrolled to achieve complete filling of the first part of thecapillary (up to the end of microfiber).

The initial starting zone was rather wide (taking into account the“dead” volume-around one half of the capillary). Then it was effectivelycompressed, in the same proportion as one could expect starting fromcross section difference. In the case of two co-axial cylinders thecross-section ratio (R=S2/S1) is: R=D2/(D2−d2), where D is the diameterof the capillary and d is the one of the microfiber inserted. By theassumptions of constant conductivity, the electric field increase in thenarrow part (E1/E2) is defined by S2/S1, and the initial zone lengthshould be narrowed in the same proportion, approximately.

The effect of observed can be combined with methods traditionally usedfor sample preconcentration. With using low conductivity buffer it waspossible to achieve an essential concentration sample increase in theplug introduced, although the peak width change was less evident.

This effect described above does not provide by itself any concentrationincrease in the introduced probe, since the volume of the sample zoneshould remain constant and the sample plug narrowing is due to it formchange. But this simple and clearly visible effect still opens a lot ofimportant applications to start with to start the separation from“initially wide” zone when it is necessary. For example, working withthe inventive technique, one can insert a microfiber into the capillaryand obtain a rather wide starting zone, with electric field applicationthe initial zone can effectively be narrowed at the end of microfiber.The latter effect, obviously, depends on the relative size of microfiberinserted, and to achieve high zone narrowing the (D−d) difference shouldbe small enough.

Solid phase microextraction and direct desorption of fluorescentlabelled analytes into the separation channel was observed. The processis monitored by the fluorescence whole column imaging detection. Theexcitation light is delivered to the separation channel using the fiber.This work successfully demonstrates the stacking process that occurs inCE coupling interface with LIF imaging detection. Based on theenhancement in fluorescence intensity, concentration efficiency can beapproximated to be as high as a 10-fold. Higher concentration efficiencycould be expected with further optimization of configuration of theinterface and the experimental conditions used, such as, dimensions ofseparation capillary and fibre, buffer concentration and appliedvoltage. The stacking effect generated by such an interface isbeneficial to separation efficiency and detection sensitivity of CEseparation.

EXAMPLE 6 Demonstration of Isotropy of Absorption and Desorption

As an alternative to conventional internal standard calibration, astandard may be loaded onto the SPME fibre prior to analysis and theloss of standard from the fibre can be monitored instrumentally. Wherethe kinetics of absorption of the internal standard analyte to the fibreis equivalent to the kinetics of desorption (binding is reversible),absorption and desorption are controlled by diffusion in the sample andthe rate of loss of standard from the fibre will be correlated withuptake of analyte by the fibre. The amount of standard lost and theamount of analyte absorbed may be measured and used to estimate theunknown concentration of analyte. Using this strategy, variation in thesample convection may be controlled for by referencing the unknownanalyte to the amount of calibrant lost from the fibre. In other words,the loss of the standard is used in this approach as an “indicator”.Extraction of an analyte onto a SPME liquid coating fibre from anagitated sample matrix is theoretically described with Eq 6 [Ai, J.Anal. Chem. 1997, 69, 1230-1236]:

$\begin{matrix}{\frac{n}{n_{0}} = \left\lbrack {1 - {\exp\left( {- {at}} \right)}} \right\rbrack} & (6)\end{matrix}$where n is the amount of extracted analyte, and n₀ is the amount ofanalyte extracted onto the fibre at equilibrium. Constant α is a measureof how fast a desorption/absorption equilibrium can be reached, and isdetermined by mass transfer coefficients, distribution coefficients,physical dimensions of the sample matrix and the fibre coating.Analogously, desorption of an analyte from a SPME liquid coating fibreinto an agitated sample matrix can be theoretically described with Eq.7:

$\begin{matrix}{\frac{Q}{q_{0}} = {\exp\left( {- {at}} \right)}} & (7)\end{matrix}$where Q is the amount of analyte remaining in the fibre and q₀ is theinitial amount of analyte extracted onto the fibre.

The isotropy of absorption and desorption of an analyte onto and from aSPME fibre is demonstrated by adding Eq. 6 and Eq. 7. The left side ofEq. 6 represents the fraction of the analyte absorbed on the fibre afterabsorption time t, while the left side of Eq. 2 represents the fractionof the analyte remaining on the fibre after desorption time t. Constantα in Eq. 6 for absorption has the same definition as constant α in Eq. 7for desorption. In other words, the value of constant α for the samecompound should be the same for both absorption and desorption under thesame conditions (sample bulk velocity and temperature). This impliesthat the sum of Q/q₀ (desorption) and n/n₀ (absorption) should be 1 atany desorption/absorption time for simultaneous absorption anddesorption of the same analyte. Our initial experimental resultsindicate that this conclusion is correct. It has been confirmed by thesimultaneous determination of the desorption time profile of deuterated(d-8) toluene and the absorption time profile of toluene: a 100 μmpolydimethylsiloxane (PDMS) fibre was loaded with deuterated toluene,then the fibre was exposed to a flowing standard toluene aqueoussolution for different times. FIG. 28 presents the values of

$\frac{Q}{q_{0}}$calculated from the desorption time profile, the values of

$\frac{n}{n_{0}}$calculated from absorption time profile, and the sum of

$\frac{Q}{q_{0}}$and

$\frac{n}{n_{0}}$for imultaneous absorption of toluene and desorption of deuterated (d-8)toluene onto and from a 100 μm PDMS fibre into water of 0.25 cm/s at 25°C. Although the sum of

$\frac{Q}{q_{0}}$and

$\frac{n}{n_{0}}$at any time is close to 1, there is a tendency for the value to beslightly smaller than 1. We ascribe this to the slight difference ofphysicochemical properties between deuterated toluene and toluene. Thedifference could be corrected by knowing the difference of thephysicochemical properties between the standard and target analytes.Experimental Details: Chemicals, Supplies, and Standard Mixtures. Allchemicals were of analytical grade. Benzene, toluene, ethylbenzene ando-xylene (BTEX) were purchased from Sigma-Aldrich (Mississauga, ON,Canada). HPLC grade methanol was purchased from BDH (Toronto, ON,Canada), and naphthalene, acenaphthene, and fluorene were purchased fromSupelco (Oakville, ON, Canada). Deuterated toluene (d-8) was purchasedfrom Cambridge isotope laboratories (Andove, Mass., U.S.A.). The SPMEholders and 100 □m polydimethylsiloxane (PDMS) fibers were obtained fromSupelco. The fibers were conditioned at 250° C. for 1 h prior to theiruse. All preparations involving toluene, ethylbenzene, and p-xylene(flammable and toxic), benzene (suspected carcinogen), naphthalene,acenaphthene, and fluorene (suspected carcinogen) were carried out in aventilated fume hood. The systems for generating the standard BTEX gasmixture and the standard BTEX and PAHs aqueous solutions have beenpreviously described (Koziel, J. A.; Martos, P. A.; Pawliszyn, J. J.Chromatogr. A 2004, 1025, 3-9).Gas Chromatography. A Varian star computer-controlled Varian 3400 CX gaschromatograph (Varian Associate, Sunnyvale, Calif.) equipped with acarbon dioxide cooled septum-equipped programmable injector (SPI) wasused for the BTEX analysis. A 0.8 mm i.d. SPI insert was coupled to aRTX-5 column (30 m, 0.25 mm i.d., 1.0 μM film thickness) and the columnwas coupled to a flame-ionization detector (FID). The injector wasmaintained at 250° C. for the PDMS fiber injection. The columntemperature was maintained at 35° C. for 2 min and then programmed at30° C./min to a maximum of 230° C. The carrier gas (helium) headpressure was set to 25 psig (˜172 kPa).

A Saturn 3800 GC/2000 ITMS system fitted with a HP-5 column (30 m, 0.25mm i.d., 0.25 μm film thickness) (Hewlett-Packard, Avondale, Pa.) wasused for the analysis of deuterated toluene and PAHs. Helium, as thecarrier gas, was set to 1 mL/min. The 1079 injector was set to 250° C.for deuterated toluene and 270° C. for PAHs, and a desorption time of 1min for deuterated toluene and 10 min for PAHs. For the analysis ofdeuterated toluene, the column temperature was maintained at 45° C. for2 min and then programmed at 20° C./min to a maximum of 180° C. For theanalysis of PAHs, the GC split valve was set to open after 5 min ofinsertion. The column temperature was maintained at 45° C. for 2 min andthen programmed at 20° C./min to a maximum of 280° C., and held for 5min. The MS system was operated in the electron ionization (EI) mode,and tuned to perfluorotributylamine (PFTBA). A mass scan from 40 to 300was acquired, and the base peak of each compound was selected andintegrated.

Discussion: This experiment discussed above (see FIG. 28) proved theisotropy of the absorption and desorption of an analyte onto and from aSPME fibre. The resulting implication is that by knowing the behaviourof either the absorption or desorption, the opposite process will alsobe understood. The practical implementation of this approach isstraightforward. To determine the concentration of an analyte in asample matrix, a known amount of isotopically labelled analogue isextracted onto a SPME liquid coating fibre. Then the fibre is exposed tothe sample matrix for a certain time, during which a part of theisotopically labeled analogue is desorbed from the fibre and a certainamount of the analyte is absorbed onto the fibre. The constant α fromdesorption allows for the calibration of absorption. This approachshould be, in principle, suitable for calibration extraction of targetcomponents from different matrices, including blood and biologicaltissues. One of the main objectives of this research program would be todemonstrate the suitability of this approach for in-vivo determinations.

In the least disruptive variant of this approach, the standards will beadded to balance the analyte loss from the matrix during extraction.This objective is accomplished by adding an amount of the standard equalto the amount of analyte being removed from the matrix. This conditionrequires a good estimation of the concentrations, but it could be usefulin investigations to verify models or previous experiments. The standardcan be an isotopically labelled analogue of the target analyte, tominimize impact on the investigated system.

In case an isotopically labelled analogue of the target analyte is notavailable, or there is more than one target analyte, a more universalapproach will be developed, based on only one reference compound loadedonto the extraction phase, and mass transfer coefficients or constants αof target analytes will extrapolated from that of the standard, based ondiffusion mass transfer [Cussler, E. L., Diffusion: mass transfer influid systems. New York: Cambridge University Press, 1997.]. However thecalibrant physicochemical properties should be similar to the analyte ofinterest to ensure that the extraction rate limited step is similar forboth compound of interest and the calibrant. FIG. 29 shows thatcalibration for extraction of fluorene works well when toluene,ethylbenzene and xylene is used, but not when benzene is used.

The isotropy of absorption and desorption of an analyte onto and from anextraction phase is independent of the geometry of the extraction phaseand the orientation of the extraction phase and sample matrix, whichallows for the use of specially designed extraction phases for somechallenging applications, such as in-vivo analysis. FIG. 30 illustratescalibration of the uptake of fluorene onto a 100 μm PDMS fiber by thedesorption of o-xylene from the same fiber into a standard PAHs aqueoussolution at a rate of 1.2 cm/s (at 25° C.), when the axis of the fiberis in different orientations to the flow direction of the standardsolution. This approach to calibration compensate for differentorientation of the extraction phase.

EXAMPLE 7 Time-Weighted Average Field Water Sampling Using Extractionwith Rod or Membrane Made of Polydimethylsiloxane (PDMS) with Calibrantin PDMS

Studies have been conducted to see if larger volume and capacitysamplers, such as PDMS rod or membrane can be used in combination withthe calibrant in the extraction phase approach. Comparing to thecommercial PDMS fiber, PDMS membrane and PDMS rod, the rod and themembrane configurations have several advantages for on-site monitoring,such as larger capacity, higher sensitivity, more flexibility and lowercost. The advantage membrane is higher rate of extraction because of thehigh surface area to volume ratio. To calibrate the environmentalfactors, such as temperature, turbulence etc., the on-rod internalstandardization method is applied, in which the standard was loaded tothe rod or membrane before introducing to the field sample. The isotropyof absorption and desorption in PDMS rod allows for the calibration ofabsorption using desorption. In the other word, the absorption of thetarget analytes can be calibrated by the desorption of the calibrantpreloaded on the rod or membrane. In the example below we discuss theperformance of the rod configuration. Membrane configuration givessimilar results with benefits of the higher rates.

Experimental: Chemicals and Supplies

All chemicals were of analytical grade. The solvent methanol wasobtained from BDH (Toronto, ON, Canada) with HPLC grade. The 100 ppmstock solution in methanol was prepared using pure solid standard ofd10-pyrene purchased from Sigma-Aldrich (US). PAHs were purchased fromSupelco (Oakville, ON, Canada). Milli-Q water was obtained bypurification and deionization of tap water immediately prior to use witha Deralpur PRO 90 CN (Seral, Germany). The pure PDMS rod was alsosupplied by Supelco which has the diameter of 1 mm. The length of 1 cm,which corresponds to about 7.85 μl of PDMS, was chosen in the currentstudy with the consideration of the length of the liner. The PDMS rodwas conditioned at 250° C. for 4 hours prior to its first use. The blankrun showed that there is no target PAHs on the rod after theconditioning. For the flow-through system, the temperature of the mixerchamber is controlled at 30° C. by using a temperature controller(Omegalux, US) to minimize the effect of the temperature on the system.

GC-MS: A Saturn 3800 GC/2000 ITMS (Varian Associate, Sunnyvale, Calif.)system equipped with a carbon dioxide-cooled septum-equippedprogrammable injector (SPI) was used for the PAHs analysis. A SPI liner(2.4 ID*4.6 OD*54 mm) with buffer coupled to a SPB-5 column (30 m, 0.25mm i.d., 0.251 μm film thickness) (Supelco, Mississauga, ON, Canada) wasused. Helium was used as the carrier gas with the flow rate of 1 mL/min.In order to put the rod into the injector, temperature program isapplied with the initial temperature at 40° C. and then increased to250° C. at a rate of 100° C./min. The column temperature was maintainedat 40° C. for 2 min and then programmed at 30° C./min to 250° C., andheld for 5 min and them programmed at 30° C./min to 280° C., and heldfor 15 min. The total run time is 30 min. The MS system was operated inthe electron ionization (EI) mode, and tuned to perfluorotributylamine(PFTBA). Mass scan from 40 to 300 was acquired, and the base peak ofeach compound was selected and integrated. The instrument was checked ona daily basis by calibration with a liquid midpoint calibrationstandard. Any deviation in area counts greater than 15% requiredre-injection of that standard; if then the deviation was still greaterthan 15% the instrument was recalibrated with a six-point calibrationplot. Peak shape quality, resolution, and retention times were alsocarefully monitored to ensure all chromatography was within all requiredspecifications.

Discussion: Standard Loading Method:

The first challenge to perform the current research is to find out asuitable standard loading method, which is fast, simple andreproducible. The headspace extraction of the calibrant dissolved insolvent or pumping oil used previously for more volatile calibrants isnot very suitable in the current study since the extraction amount wouldbe too low and the extraction time would be extremely long to obtainenough loading amount due to the low volatility of PAHs. Development ofan appropriate method was performed using deuterated pyrene as thestandard for loading. To load the appropriate amount of deuteratedpyrene with high reproducibility, the loading was performed by puttingthe rod directly in the standard solution with agitation in this study.The extraction time was set as half an hour and the reproducibility wasgood which was lower than 7%. Three rods were involved in this study andthe RSDs for the extraction by all the three rod are listed in Table 3.The amount of the standard loaded onto the rod can be also adjusted bychanging the concentration of standard loading solution and extractiontime.

Absorption of the PDMS rod:

In order to determine the sample concentration, q₀ need to be clarifiedbefore the sampling. To perform the extraction, 10 ppb standard solutionwas prepared by spiking 1 μl 100 ppm deuterated pyrene (calibrant) into10 ml deionized water containing in 10 ml vial. The rod was introducedinto the vial with a stir bar stirring at 1000 rpm. After certainextraction time, the rod was taken out form the solution with tweezers,dried with lint-free tissue and then immediately transferred to the GCinjector for analysis. It was found that the extracted mass ofdeuterated pyrene increases linearly initially and reached stable levelafter extracting for more than 2 hrs. Comparison with the results ofdirect syringe injection of 1 μl 100 ppm pyrene-d10 into the GC injectorindicates that the extraction is an exhaustive extraction due to thehigh capacity of the rod.

Isotopic of the Absorption of the Analytes and Desorption of theStandard:

Experiments were conducted to validate the existence of the isotopic ofthe absorption of the analytes and the desorption of the standard. Theexperiment involved the simultaneous determination of the desorptiontime profile of deuterated pyrene as the calibrant and the absorptiontime profile of pyrene as the component. The rod was preloaded withdeuterated pyrene and then exposed to the flow-through system withdifferent exposure time. FIG. 31 shows the value of

$\frac{q}{q_{0}}$calculated from the resulting desorption time profile, the value of

$\frac{n}{n_{0}}$calculated from the resulting absorption time profile, and the sum of

$\frac{q}{q_{0}}$and

$\frac{n}{n_{0}}$at any time is close to 1, which demonstrate the isotopic of theabsorption and desorption for long times (100 h). The isotropy of theabsorption and desorption on the rod allows the calibration ofabsorption of the analytes by the desorption of the standard. Thisapproach is especially important for the calibration of on-siteanalysis. In particular if the thick extraction phase is used thisexample demonstrates that the desorption of the calibrant from highcapacity extraction phase can persist for long period of time, whichfacilitate calibration of long exposure times including TWA sampling.The concentrations of PAHs in the flow-through system were determined byusing PDMS rod extraction and on-rod internal standardization method.The concentrations determined by this new calibration approach agreewell with the results obtained from external calibration method, whichdemonstrated the feasibility of this approach to the field watersampling.

EXAMPLE 8 Equilibrium in-Fibre Standardisation Technique for Solid-PhaseMicroextraction

This example describes an exemplary solid phase microextraction using astandard loaded into the fibre coating as a means of internalstandardisation for the analysis of samples contained in vials.Reproducible amounts of standards were loaded into a SPME non-porousfiber. It was found that spiking a few microliters of liquid standardssuch as benzene, toluene, ethylbenzene, and xylenes (BTEX) and/or addinga few milligrams of solid standards such as naphthalene into few gramsof pump oil sealed in a 20 milliliter vial provided an excellentstandard generator, and allowed for reproducible loading of standards(RSD<4%) up to hundreds of times. When standards were introduced into asample vial together with a fiber, extraction of analytes into the fiberand desorption of the standards into the sample matrix occuredsimultaneously. Quantification was then based on the equilibriumdistribution of the standards and the analytes between the fibre coatingand the sample matrix in the vial. A comparison of equilibrationprofiles obtained using traditional internal standardisation and thein-fibre approach generally showed the same equilibration behaviour.This method, according to one aspect of the invention was successfullyused to correct for matrix effects in the BTEX analysis of a winesample.

Introduction. Internal standardisation is a well known calibrationapproach in analytical chemistry that is used to improve the accuracyand precision of experimental data to account for such factors as samplematrix effects, losses during sample preparation, and irreproducibilityin such parameters as sample injection in GC (Haefelfinger, Chromatogr,1981, 218, 73-81). Solid-phase microextraction (SPME) as a samplepreparation and extraction technique is no exception, with internalstandardisation often used for quantification particularly whenanalysing complex samples. However, the addition of an internal standardprovides an additional step in sample preparation. For completelyautomated analysis, two robotic arms are required, one to provide thestandard spike and the other for SPME. There are also situations inwhich the addition of an internal standard is not practical, such ason-site or in-vivo applications.

SPME is a solvent free technique designed for rapid sampling and samplepreparation. (Pawliszyn, J. Solid Phase Microextraction Theory andPractice; Wiley-VCH: Chicester, 1997). The most common form of thetechnique uses a fibre coated with a liquid polymeric film, which isexposed to the sample, extracting analytes from it until equilibrium isreached. The amount of analyte absorbed by the coating at equilibrium(n_(f)) is linearly proportional to the initial concentration in thesample (C₀) by eq 8:

$\begin{matrix}{n_{f} = {\frac{K_{fs}V_{f}V_{s}}{{K_{fs}V_{f}} + V_{s}}C_{0}}} & (8)\end{matrix}$where K_(fs) is the fibre/sample distribution coefficient, V_(f) is thevolume of the fibre coating and V_(s) is the volume of the sample. Foranalysis in a vial containing headspace this equation should beexpressed as shown in eq 9:

$\begin{matrix}{n_{f} = {\frac{K_{fs}V_{f}V_{s}}{{K_{fs}V_{f}} + {K_{hs}V_{h}} + V_{s}}C_{0}}} & (9)\end{matrix}$where K_(hs) and V_(h) represent the headspace/sample distributioncoefficient and the volume of the headspace, respectively.

The fact that SPME is an equilibrium rather than an exhaustiveextraction technique means that even after the extraction process hasbeen completed a substantial portion of the analytes usually remain inthe matrix. This presents an opportunity for quantification based oninternal standardisation, namely that the standard is loaded onto thefibre prior to the extraction step, instead of spiked into the sample.Example 6 have explored the kinetics of the technique, demonstratingthat the absorption and desorption processes are isotropic, which allowsfor calibration of the rate of absorption using the rate of desorption.

The current example aimed to expand on this technique to fundamentallyassess the in-fibre standardisation approach with systems reachingequilibrium. The in-fibre standardisation approach was developed forautomated sampling from milliliter quantities of liquids in vials andused for the analysis of BTEX in a wine sample.

Theoretical Considerations. The equilibrium equation for SPME, mostgenerally described by eq 7 is derived from the knowledge that theamount of analyte in the system will remain the same before and afterthe extraction. This mass balance equation can therefore be expressed byeq 10:n _(T) =n _(f) +n _(h) +n _(s)  (10)where n_(T) is the total number of moles of analyte in the system, andthe remaining terms denote the amount of the analyte in the fibre,headspace and sample respectively at equilibrium. Using this form ofexpressing the mass balance, leads to eq 11:

$\begin{matrix}{n_{f} = {\frac{K_{fs}V_{f}}{{K_{fs}V_{f}} + {K_{hs}V_{h}} + V_{s}}n_{T}}} & (11)\end{matrix}$

From this equation, it is apparent that no matter where the standard oranalyte of interest starts in the system, at equilibrium the amount inthe fiber should be the same.

A further consideration is the kinetics of the process. For traditionalSPME the kinetics for both direct and headspace extraction can bedescribed by eq 12 (Ai, J. Anal. Chem. 1997, 69, 3260-3266; Ai, J. Anal.Chem. 1997, 69, 1230-1236):

$\begin{matrix}{\frac{n}{n_{f}} = {1 - {\exp\left( {- {at}} \right)}}} & (12)\end{matrix}$where n is the moles of analyte in the coating at time t, a is aconstant that is dependant on the volumes of the fibre, headspace andsample, mass transfer coefficients, distribution coefficients and thesurface area of the fibre. The kinetic processes involved for desorptionof analytes from the fibre coating is defined by eq 13:

$\begin{matrix}{q = {n_{0}{\frac{V_{s}}{{K_{fs}V_{f}} + V_{s}}\left\lbrack {1 - {\exp\left( {- {at}} \right)}} \right\rbrack}}} & (13)\end{matrix}$where q is the moles of the analyte lost from the coating at time t andn₀ represents the moles of the compound originally loaded. For the caseof in-vial analysis the moles remaining on the fibre (n) at time t canbe expressed asn=n ₀ −q  (14)

From eqs 13 and 14 it is apparent that

$\begin{matrix}{n = {n_{0} - {n_{0}\frac{V_{s}}{{K_{fs}V_{f}} + V_{s}}} + {n_{0}\frac{V_{s}}{{K_{fs}V_{f}} + V_{s}}{\exp\left( {- {at}} \right)}}}} & (15)\end{matrix}$However, as the exponential term disappears as time goes to infinity,therefore

$\begin{matrix}{n_{f} = {n_{0} - {n_{0}\frac{V_{s}}{{K_{fs}V_{f}} + V_{s}}}}} & (16)\end{matrix}$Substituting eq 16 into eq 15 and rearranging gives

$\begin{matrix}{\frac{n - n_{f}}{n_{0} - n_{f}} = {\exp\left( {- {at}} \right)}} & (17)\end{matrix}$Comparing eq 17 with eq 12, it can be concluded that for in-vialanalysis the isotropy of absorption and desorption of an analyte fromthe fibre still maintains. A similar expression can be derived forheadspace analysis with a suitable adjustment in the definition of a.

Experimental: Materials. Ethylbenzene-d₁₀ (99+%), ethyl benzene,o-xylene (98%, HPLC grade), naphthalene (99+%, scintillation grade) andcarbon disulfide (99.9+%, HPLC grade) were purchased from Sigma-Aldrich(Milwaukee, Wis., USA). Benzene (analytical reagent) was from BDH Inc.(Toronto, ON, Canada), toluene (Guaranteed Reagent) from EMD (Gibbstown,N.J., USA), D₈-Naphthalene (99%) from Cambridge Isotope LaboratoriesInc. (Andover, Mass., USA). HPLC grade methanol was obtained from FisherScientific (Nepean, ON, Canada), the vacuum pump oil was supplied by BOCEdwards (Wilmington, Mass., USA), and the poly(dimethylsiloxane) (PDMS)membrane material was supplied by Specialty Silicone Products Inc.(Ballston Spa, N.Y., USA). PDMS (100 μm) SPME fibres and Tenax (TA80/100mesh) were purchased from Supelco (Bellefonte, Pa., USA). Water purifiedfrom a Barnstead ultrapure water system (Dubuque, Iowa USA) was usedthroughout. All gases were supplied by Praxair (Kitchener, ON, Canada)and were of ultra high purity. Ten or twenty milliliter sample vialswere used for automated analysis with magnetic crimp caps and PTFEcoated silicone septa (Microliter Analytical Supplies, Suwanee, Ga.,USA). The dry white wine sample was obtained from a local liquor store.

GC Analysis. Gas chromatography was performed on a Varian™ (Mississauga,ON, Canada) 3800 gas chromatograph coupled with a Saturn™ 2000 MS systemcontrolled by computer using Varian Saturn Workstation software (ver.5.51) or with a FID detector using Star Chromatography Workstation (ver5.51). Automated analysis was performed using a CTC CombiPal™autosampler (Zwingen, Switzerland) using the associated Cycle Composer™software (ver 1.4.0). The PAL was equipped with a SPME fibre holder, atemperature controlled six vial agitator tray and a fibre conditioningdevice. Separation was performed using a 30 m×0.25 μm×0.25 mm I.D.Rtx-5MS fused silica column from Restek (Bellefonte, Pa., USA). Foranalysis of BTEX the column was initially set at 40° C. for 4 minutesand then ramped at 15° C./min to 130° C. giving a total run time of 10minutes. The injector was set at a temperature of 250° C. and helium wasused as the carrier gas at a flow rate of 1 mL/min. For analysis ofnaphthalene, the column was initially set at 40° C. for 1 min and thenramped at 20° C./min to 220° C. giving a total run time of 10 min. Thetemperature of the injector was set at 250° C. and helium was used asthe carrier gas at a constant pressure of 12 psi. For both analytes a 1minute desorption time in the GC injection port was used, which wasimmediately followed by a 2 minute bake-out at 250° C. in theautosampler fibre conditioning device.

FID was used at a temperature of 300° C. with gas flows for hydrogen,high purity air and make-up gas (nitrogen) set at 300, 30 and 25 ml/minrespectively. For the mass spectrometry detection experiments, electricionisation was used with temperatures of 170, 50 and 260° C. for thetrap, manifold and transfer line respectively. A scan range of 70 to 125m/z was used and quantification was performed using 78 m/z for benzene,91 for toluene, 98 and 116 for deuterated ethyl benzene and 91 and 106for ethyl benzene and o-xylene. For naphthalene, a scan range of 100 to160 m/z was used and quantification was performed using 128 m/z fornaphthalene and 136 m/z for deuterated naphthalene.

For the automated analysis a sampling temperature of 35° C. was used.The internal standard was loaded onto the fibre by exposure to theheadspace of a 20 mL sample vial containing 4.00 g of vacuum pump oilspiked with deuterated ethyl benzene at a concentration of 0.47 mg/g.The loading time was 1 minute with an agitation speed of 500 rpm. Thefibre was then immediately exposed to the headspace of a 10 mL vialcontaining the sample for 5 minutes, again using a 500 rpm agitationspeed. The sample volume used unless otherwise specified in theseexperiments was 3.0 mL. A 6 minute pre-extraction equilibration of thesample was performed in the agitation unit at 500 rpm. For loading ofnaphthalene, a 2.00 g solution pump oil was used containing 2.0 mg/gnaphthalene. All other conditions were the same as in the ethyl benzeneexperiments unless otherwise specified.

Addition of Standard Spike to Vials. Standards used for constructingcalibration curves and sample “spikes” to test the method were preparedby spiking the sample with a standard of the target compounds preparedin methanol. Initially this was done after the vial had been capped bymeans of a 10 or 100 μL syringe. However, using this approach a steadydecline in peak areas for the analytes was observed that was related tothe amount of time between spiking and sampling. The decline was worsewith ethyl benzene and xylene than with benzene and toluene. Thissuggested the behaviour was caused by absorption of the compounds intothe small part of the vial septum silicone layer exposed throughaddition of the standard spike. To overcome this difficulty it wasnecessary to spike the solutions prior to capping. To minimiseevaporation it was necessary to add the spike below the level of thesolution in the vial, a similar approach to that adopted for standardpreparation in EPA method 5021 A (US Environmental Protection Agency,Method 5021A: Volatile Organic Compounds in Various Sample MatricesUsing Equilibrium Headspace Analysis, 2003).

Results and Discussion: Internal Standard Loading on Fiber. The firstchallenge was to find a method that would allow, automated, fast andreproducible loading of the standard into the fiber. Development of anappropriate method was performed using ethyl benzene as the “standard”for loading. Sampling from the headspace of a vial containing pure ethylbenzene resulted in unmanageably high loading on the fiber coating evenfor extremely short absorption times. This was true even when cooled to5° C. in the sample tray. The use of diluted solutions of ethyl benzenein water to reduce the loading to an acceptable level showed that themass of ethyl benzene withdrawn from the vial during each loading stepwas a significant percentage of the total. This made it impossible toreuse a “loading” vial, which is not practical in terms of the number ofstandard solution vials required for a automated sample list. The use ofalternative techniques, such as vials containing ethyl benzene absorbedonto Tenax, or PDMS membrane showed similar problems. Injecting theneedle into a headspace of a vial containing pure ethyl benzene, but notexposing the fiber coating showed a workable and reproducible loading,except the needle sometimes being blocked with a piece of septum.

Finally, a system was adopted whereby the ethyl benzene was dissolved invacuum pump oil, to reduce the K_(fs) partition coefficient for thestandard into the fiber. Using this method gave an acceptable andreproducible loading with 1 min exposure to the standard solutionheadspace. This also worked well for naphthalene. The amount loaded intothe fiber can be further adjusted by spiking different amount of thestandard into the vacuum pump oil and/or exposing the fiber fordifferent time. Using this approach, each loading cycle only withdrew0.0087% of the ethyl benzene in the vial, making it possible to use thevial for at least 115 injections before 1% of the vial contents had beenremoved. Reproducibility of the loading step for ethyl benzenedetermined by FID was 1.9% for 40 injection cycles, whilst the value was2.6% for loading followed by equilibration with a vial containing 3 mLof water for 10 minutes in 20 injection cycles. For naphthalene loadingreproducibility was 2.0% for 30 injection cycles, whilst the value was3.6% for loading followed by equilibration with a vial containing 3 mLwater for 10 min in 20 injection cycles. In theory, the standardsolution can be used at least 300 times before 1% of the vial contentshad been removed.

Comparison of Equilibration Curves. As a first investigation of theapproach, equilibration profiles using traditional SPME and in-fiberstandardisation SPME for ethyl benzene and naphthalene were comparedwhen exposed to empty 10 mL headspace vials, (actual volume wasdetermined to be 11.5 mL). The two processes were also investigatedusing direct immersion of the fiber in vials filled with water andheadspace experiments in vials containing 3 mL of water. Under theconditions studied the equilibration time was not influenced by thelocation of the standard at the beginning of the equilibration process.

Applications to Real Sample Matrices. To test the in-fibrestandardisation method, the technique was used to examine BTEX in spikedwhite wine with MS detection. The method was linear for BTEX compoundsover the tested range of 0.09-73 μg/L. For all of the compounds, thelinearity was higher than 0.9998 in both the calibration curves and thecurves normalized by internal standard. The recoveries from wine spikedwith 7.3 μg/L BTEX, calculated using external calibration and thein-fibre standardisation approach are given in Table 2. Deuterated ethylbenzene was used as the internal standard.

TABLE 2 Recoveries for BTEX in White Wine Recovery Recovery calculatedusing calculated using internal standard in the fibre Compound externalcalibration (%) (%) Benzene 81.2 ± 5.2 102.3 ± 5.8 Toluene 83.2 ± 0.7105.1 ± 0.2 Ethyl Benzene 76.8 ± 1.0  99.3 ± 0.6 o-Xylene 72.1 ± 1.5 91.2 ± 1.3

It was demonstrated that the in-fibre technique gives improved recoveryfor the determination of these compounds than seen using externalcalibration. With d₁₀-ethylbenzene as internal standard, 99% recoverywas obtained for ethylbenzene. The slightly higher deviations from 100%recovery for the other analytes can largely be attributed to differencesin the interactions of these compounds with the matrix compared to theinternal standard.

Conclusions. From this example, it is confirmed that the in-fibrestandardisation approach works successfully under equilibrium conditionsand can be easily automated. The developed procedure requires only asingle arm autosampler, unlike SPME with traditional internalstandardisation that requires a dual arm system. Equilibration time wasnot affected by where the standard commenced in the system. Thetechnique was applied to the analysis of BTEX in wine, successfullycorrecting for matrix effects. This method can also be used with otherequilibration extraction techniques, such as liquid phasemicroextraction (LPME) or membrane extraction.

EXAMPLE 9 Standards in Liquid Phase for Liquid-Phase Microextraction

This example describes an exemplary liquid phase microextraction (LPME)using a standard loaded into extraction liquid as a means of internalstandardisation for the analysis of samples contained in vials.Information about LPME techniques can be found in following references:E. Psillakis and N. Kalogerakis. Developments in single-dropmicroextraction. Trends Anal. Chem. 2002, 21, 54-64 and K. E. Rasmussenand S. Pedersen-Bjergaard Developments in hollow fiber-based,liquid-phase microextraction. Trends Anal. Chem. 2004, 23, 1-10. Thekinetics of the absorption and desorption of analytes for a variant ofLPME single-drop headspace liquid-phase micoextraction (SD-HS-LPME) werestudied. Procedures used in other variants of the LPME technique areanalogues. It was found that the desorption of calibrant from theextraction phase into sample matrix is isotropic to the absorption ofanalyte (component) from the sample matrix into the extraction phaseunder the same conditions, which allows for the calibration ofabsorption using desorption. The calibration was accomplished byexposing the extraction phase, pre-added with a standard, to the samplematrix. The information from the desorption of standard, i.e., timeconstant α, could be directly used for estimating the concentration ofthe target analyte in the sample matrix. The developed kineticscalibration method of headspace LPME was successfully used to correctthe matrix effects in the BTEX analysis of orange juice sample. In thisstudy, headspace LPME technique, for both static and dynamic, wassuccessfully automated by using a CTC CombiPal autosampler. Alloperations of headspace LPME, include sample transfer and agitation,filling of extraction solvent, exposing the solvent in the headspace,withdrawing the solvent to syringe and introducing the extraction phaseinto injector, were auto performed by the CTC autosampler. The fullyautomated headspace LPME technique is more convenient and improves theprecision and sensitivity. This automated headspace LPME technique canbe also used to obtain the distribution coefficient between the samplematrix (aqueous or other solution) and the extraction phase (1-octanolor other solvent). The distribution coefficient between 1-octanol andorange juice at 25° C. was obtained by using this technique.

Chemicals and Supplies. All chemicals were of analytical grade. Benzene,d₆-benzene, toluene, d₈-tuluene, ethylbenzene, o-xylene, 1-octanol(HPLC, 99+%) were from Sigma-Aldrich (Mississaga, ON, Canada). HPLCgrade methanol was purchased from BDH (Toronto, ON, Canada). HamiltonModel 701N 10 μL syringes (26s gauge, no. 2 point style bevel tip) werepurchased from Hamilton (Reno, Nev., USA). 10 mL screw vials withmagnetic crimp caps and PTFE coated silicone septa (Supelco, Oakville,ON, Canada) were used for automated analysis. Water purified from aNanopure filter (Barnstead, Dubuque, Iawa, USA) was used throughout.Ultra high purity helium was supplied by Praxair (Kitchener, ON,Canada). The orange juice sample was purchased from a local supermarket.

Instrument. A Saturn 3800 GC/2000 ITMS system fitted with a SPB-5 column(30 m, 0.25 mm i.d., 0.25 μm film thickness) (Supelco, Mississauga, ON,Canada) was used for the analysis of BTEX. Helium as the carrier gas wasset to 2 mL/min. The column temperature was maintained at 80° C. for 1min and then programmed at 20° C./min to 120° C., and then programmed at50° C./min to 250° C., and held for 4.4 min. The total run time was 10min. An i.d. 2 mm liner packed with glass wool was used for the 1079injector. The injector was set to 250° C. with a split ratio of 10:1.The syringe will hold 10 second in the injection after sample wasinjected. The MS system was operated in the electron ionization (EI)mode, and tuned to perfluorotributylamine (PFTBA). The EI was set toturn on at 1 min and turn off at 3 min (before the elution of solvent).A mass scan from 40 to 120 was used and quantification was performedusing m/z 78 for benzene, m/z 84 for d₆-benzene, m/z 98 for d₈-tolueneand m/z 91 for toluene, ethylbenzene, and o-xylene.

Extraction and desorption procedure. All extraction procedure wasperformed with a CTC CombiPal autosampler (Zwingen, Switzerland) usingthe associated Cycle Composer software (ver 1.4.0). An 870 ppm stocksolution of BETX components was prepared in methanol. 870 ppb standardsolutions of BTEX were prepared daily by spiking the stock solution topure water with a CTC autosampler. To avoid the effect of time, thesolution was spiked prior to capping and the spike was added below thelevel of the water in the vial.

The extraction of analytes in the sample and the desorption of internalstandard were performed in a 10 mL vial, containing 3 mL 870 ppb BTEXaqueous solution, for the determination of adsorption profile, or 3 mLpure water, for the determintion of desorption profile. The single-dropextraction phase were 1 μL 1-octanol, for the determination ofadsorption profile, or 1 μL 1-octanol containing 870 ppm internalstandard (BTEX or d₆-benzene and d₈-toluene), for the determination ofdesorption profile.

For static SD-HS-LPME, the 10 mL sample vial was transferred from sampletray to the vortex agitator with temperature controller, shaking 2 minat 500 rpm, then the Hamilton 701 10 μL syringe, filled with 1 μL1-octanol, pierced the septum and slowly exposed the 1 μL 1-octanol (0.1μL/s) in the headspace of sample vial. After different extraction time,the 1 μL 1-octanol was slowly withdraw to the barrel (0.1 μL/s) and wasintroduced to GC/MS to analysis.

For dynamic SD-HS-LPME, the sample vial was also shook 2 min at 500 rpmin the vortex agitator, then the Hamilton 701 10 μL syringe, filled with1 μL 1-octanol, pierced the septum and the plunger was slowly depressedto expose the 1 μL 1-octanol (0. 1 μL/s) in the headspace of sample vialand then the 1 μL 1-octanol were immediately withdrew to the barrel ofthe syringe. The sample vial was then shook 10 second at 500 rpm, the 1μL 1-octanol was exposed and withdrew again. This procedure was repeateddifferent times before the 1 μL 1-octanol were introduced to GC/MS.(Caution: due to the needle of the syringe will shaking with sample vialwhen perform the dynamic operation, the dynamic program must to becarefully set, otherwise the needle of syringe will be easy damaged. Thebest program is like the operation of CTC autosampler for headspaceSPME. The syringe can be used hundreds times still keep good conditionif the program was optimized.)

Quantification. The solvent for preparing standard solution willobviously affect the sample transfer into the column of GC, especiallythe standard solution was introduced to high temperature injector. Inthis study, it was found that the peak areas of BTEX for 870 ppmmethanolic solution, compared with the peak areas of BTEX for 870 ppm1-octanolic solution, were just about 50%. To avoid the effect ofsolvent for the quantification, the standard solutions for calibrationalso were prepared with 1-ocatanol. Good precision (RSD<5%) andlinearity (R²>0.999) were obtained for the calibration curves.

Analysis of BTEX in Orange Juice. The recoveries from orange juicespiked with 870 ppb BTEX calculated against with external calibration(standards prepared in water) and kinetics calibration approach (timeconstant α was determined at first, and theexposition-withdrawal-agitation procedure was repeated 5 times whenperform the determination of real sample) are given in Table 3. Theresults demonstrate that the kinetic calibration technique gives moreaccurate determination of these compounds than external calibration.

TABLE 3 Calculated recoveries of BTEX from orange juice with and withoutcalibration with single drop extraction. Relative recovery (%) (RSD, %;n = 3) Using external Using kinetic Compound calibration calibrationBenzene 121 (5.3)  99 (5.8) Toluene 97 (4.6) 94 (3.5) Ethylbenzene 73(5.7) 91 (5.6) o-Xylene 73 (6.3) 95 (3.5)

EXAMPLE 10 Membrane Extraction with Calibrant in the Stripping Fluid

This example describes a new technique for calibration in membraneextraction processes, by adding an analytically non-interfering internalcalibrant in the stripping fluid. Description of membrane techniques canbe found in the following reference: Jönsson, J. {dot over (A)}.,Mathiasson, L. J Chromatogr. A 2000, 902,205-225. A membrane extractionwith sorbent interface (MESI) system was used to evaluate this approach.Description of membrane techniques can be found in the followingreference: Lord, H., Yu, Y., Segal A., Pawliszyn J. Anal. Chem. 2002,74, 5650-5657. During membrane extraction, the internal calibrant fromwithin the carrier (stripping) gas and the target analyte from thesample matrix will permeate simultaneously through the membrane inopposite directions. The change of the accumulation amounts of internalcalibrant in the microtrap can be used as a means of calibration tocorrect for the variation of the extraction rate due to variableenvironmental factors, such as the sample feed velocities and thetemperature of the membrane. Thus, this approach should allow for moreaccurate estimates of target analyte concentrations for complicatedsampling conditions.

Theory: The permeation of analytes through a nonporous polymer membraneis generally described in terms of a “solution-diffusion” mechanism,which consists of seven consecutive steps (FIG. 32). Permeation ratesare independent of whether the receiving chamber contains a vacuum or agas other than the penetrant. Therefore, the internal calibrant, fromthe carrier (stripping) gas, and the target analyte, from the samplematrix, will permeate simultaneously through the membrane in oppositedirections. When the diffusion reaches a steady state, these processesfollow Fick's first law of diffusion.

For the internal standard:

$\begin{matrix}{{J_{I} \equiv {\frac{1}{A}\frac{\mathbb{d}n_{I}}{\mathbb{d}t}}} = {{{- D_{c,I}}\frac{\mathbb{d}C^{c,I}}{\mathbb{d}x}} = {{{- D_{m,I}}\frac{\mathbb{d}C^{m,I}}{\mathbb{d}x}} = {{- D_{s,I}}\frac{\mathbb{d}C^{s,I}}{\mathbb{d}x}}}}} & (18)\end{matrix}$Where J_(I) is the mass flux of the internal calibrant from the carrier(stripping) gas to the sample matrix; A is the surface area of themembrane; dn_(I) is the amount of the internal calibrant that permeatedfrom the carrier (stripping) gas during the time period dt; D_(c,I)D_(m,I) and D_(s,I), are diffusion coefficients of the internalcalibrant in the carrier (stripping) gas, membrane, and sample matrix,respectively, and C^(c,I), C^(m,I), and C^(s,I) are concentrations ofthe internal calibrant in the carrier (stripping) gas, membrane, andsample matrix, respectively.A linear concentration gradient in the boundary layers and membrane isassumed:

$\begin{matrix}{{J_{I} \equiv {\frac{1}{A}\frac{\mathbb{d}n_{I}}{\mathbb{d}t}}}\mspace{25mu} = {{{- \frac{D_{C,I}}{\delta_{1,I}}}\left( {C_{1} - C_{0}} \right)}\mspace{25mu} = {{{- \frac{D_{m,I}}{\delta_{m,I}}}\left( {C_{3} - C_{2}} \right)}\mspace{25mu} = {{- \frac{D_{s,I}}{\delta_{2,I}}}\left( {C_{5} - C_{4}} \right)}}}} & (19)\end{matrix}$Where δ_(1,I), δ_(m,I) and δ_(2,I) are the thickness of the insideboundary layer, the membrane, and the outside boundary layer,respectively; C₀ is the concentration of the internal calibrant in thebulk of the carrier (stripping) gas; C₁ is the concentration of theinternal calibrant in the inside boundary layer at the interface of themembrane and the inside boundary layer; C₂ is the concentration of theinternal calibrant in the membrane at the interface of the membrane andthe inside boundary layer; C₃ is the concentration of internal calibrantin the membrane at the interface of the membrane and the outsideboundary layer; C₄ is the concentration of the internal calibrant in theoutside boundary layer at the interface of membrane and the outsideboundary layer; and C₅ is the concentration of the internal calibrant inthe bulk of the sample matrix. The mass transfer coefficients of theinternal calibrant in the inside boundary layer h_(c,I), membraneh_(m,I), and outside boundary layer h_(s,I) are defined as

$\begin{matrix}{{h_{c,I} = \frac{D_{c,I}}{\delta_{1,I}}},{h_{m,I} = \frac{D_{m,I}}{\delta_{m,I}}},{{{and}\mspace{14mu} h_{s,I}} = {\frac{D_{s,I}}{\delta_{2,I}}.}}} & {{Equation}\mspace{20mu} 19}\end{matrix}$can then be written in the forms:J=−h _(c,I)(C ₁ −C ₀)  (20)J=−h _(m,I)(C ₃ −C ₂)  (21)J=−h _(s,I)(C ₅ −C ₄)  (22)Assuming a quick partition equilibrium exists at the interfaces ofmembrane and the gas boundary layer:

$\begin{matrix}{K_{I} = {\frac{C_{2}}{C_{1}} = \frac{C_{3}}{C_{4}}}} & (23)\end{matrix}$Where K_(I) is the distribution coefficient of the internal calibrantbetween the membrane and the gas phase.Substitution of Equation 8 into Equation 6 yields:J=−h _(m,I) K _(I)(C ₄ −C ₁)  (24)Combining and rearranging Equations 5, 6, and 9 leads to:

$\begin{matrix}{J_{I} = {{\frac{1}{\left( {\frac{1}{h_{c,I}} + \frac{1}{h_{s,I}} + \frac{1}{K_{I}h_{m,I}}} \right)}\left( {C_{0} - C_{5}} \right)} = {h_{{total},I}\left( {C_{0} - C_{5}} \right)}}} & (25)\end{matrix}$Where h_(total,I) is the total mass transfer coefficient for theinternal calibrant, and it is defined as

$\begin{matrix}{\frac{1}{h_{{total},I}} = {\frac{1}{h_{c,I}} + \frac{1}{h_{s,I}} + \frac{1}{K_{I}h_{m,I}}}} & (26)\end{matrix}$Using the same procedure, the flux equation for analyte in the samplematrix into the carrier (stripping) gas can be deduced as:

$\begin{matrix}{J_{s} = {{\frac{1}{\left. {\frac{1}{h_{c,s}} + \frac{1}{h_{s,s}} + \frac{1}{K_{s}h_{m,s}}} \right)}\left( {C_{a} - C_{f}} \right)} = {h_{{total},s}\left( {C_{a} - C_{f}} \right)}}} & (27)\end{matrix}$Where h_(c,s), h_(m,s), and h_(s,s) are the mass transfer coefficient ofthe target analyte in the inside boundary layer, membrane, and outsideboundary layer, respectively. K_(s) is the distribution constant of thetarget analyte between the membrane and the gas phase. C_(a) is theconcentration of target analyte in the bulk of the sample matrix; C_(f)is the concentration of the target analyte in the bulk of the carrier(stripping) gas. h_(total,s) is the total mass transfer coefficient forthe target analyte and it is defined as

$\begin{matrix}{\frac{1}{h_{{total},s}} = {\frac{1}{h_{c,s}} + \frac{1}{h_{s,s}} + \frac{1}{K_{s}h_{m,s}}}} & (28)\end{matrix}$

h_(c,s), h_(s,s), h_(c,I) and h_(s,I) are mass transfer parameters thatreflect the effects of the boundary layer between the two sides of themembranes. As a simplified model, for the sample feed or the carrier(stripping) gas extraction side, gas can be regarded as a laminar flowpast the flat plate and the following equation can be used to quantifythe mass transfer through the boundary layer.

$\begin{matrix}{h = {0.626\frac{D}{L}\left( \frac{{Lv}^{0}}{\upsilon} \right)^{1/2}\left( \frac{\upsilon}{D} \right)^{1/3}}} & (29)\end{matrix}$Where D is the diffusion coefficient of the analyte being transferred; Lis the membrane effect length; v⁰ is the bulk velocity of the sample orcarrier (stripping) gas; and ν is the kinematic viscosity.

In most cases, C₅ and C_(f) remained negligibly small during theexperiment. This is because (1) the amounts of penetrant (internalcalibrant or target analyte) were very small, (2) the carrier(stripping) gas continuously purged the inner surface of the membrane toprevent an increase in penetrant concentration near the inner surface ofthe membrane, and the penetrant of the calibrant was immediatelyreleased to the surrounding sample matrix, minimizing the possibility ofaccumulation on the outer surface of the membrane.

Thus, Equations 25 and 27 can be simplified as:

$\begin{matrix}{J_{I} = {{\frac{1}{A}\frac{n_{I}}{t}} = {h_{{total},I}C_{0}}}} & (30)\end{matrix}$

$\begin{matrix}{J_{s} = {{\frac{1}{A}\frac{n_{s}}{t}} = {h_{{total},s}C_{a}}}} & (31)\end{matrix}$

If r is defined as

$\begin{matrix}{r = \frac{h_{{total},s}}{h_{{total},I}}} & (32)\end{matrix}$and Equation 31 is divided by Equation 15, the target analyteconcentration can be expressed as

$\begin{matrix}{C_{a} = {{\frac{n_{s}}{n_{I}r}C_{0}} = {\frac{f_{s}H_{s}}{f_{I}H_{I}r}C_{0}}}} & (33)\end{matrix}$

In Equation 33, C₀ is the known concentration of the internal standardin the carrier (stripping) gas; r can be calibrated based on differentscenarios, which is addressed below. n_(s) is the extracted amount ofthe target analyte in the microtrap. n_(I) is the permeated amount ofinternal calibrant in the matrix which is a difference between theaccumulation of internal calibrant with and without membrane module.During calibration in the lab, the amount of penetrated internalcalibrant can be conveniently measured using the SPME-GC systemdiscussed as below. Both f_(s) and f_(I) are GC calibration factors forthe target analyte and internal calibrant, respectively, and H_(s) andH_(I) are the peak areas of the target analyte and the internalcalibrant, obtained individually by GC analysis.

Experimental details: MESI-portable GC with Internal Calibrant System. Ageneral scheme of the MESI-portable GC internal calibrant system hasbeen designed. The main components of this system include the membranemodule, the microtrap (sorbent interface), the control unit of thecooler and heater, the gas chromatograph, and a silanized Pyrex glasscylinder with a permeation tube for the production of a stable flow ofchemical vapour as the internal calibrant. The design of the membranemodule, microtrap, and control unit of the cooler and heater have beenpresented in previous work. The PDMS membrane was mounted inside themodule and the effective membrane surface area was 3.63 cm². Microtrapsfor this study were made from a small diameter silicosteel® coatedtubing (5 cm×0.75 mm i.d.) packed with PDMS material. The control unitkept the microtrap at −5° C. during preconcentration and heated to 200°C. during injection procedure. A Model 08610 SRI portable gaschromatograph (SRI Instruments, Torrance, Calif., USA) was, equippedwith a FID detector and a MXT-Volatiles column (10 m×0.53 mm i.d., with3.0 μm phenylmethylpolysiloxane film) (Restek Corp., Bellefonte, Pa.,USA). The detector temperature was maintained at 250° C. The initialcolumn temperature was set at 80° C., held for 10 minutes, and thenincreased at a rate of 20° C./min to 180° C. Nitrogen was used as thecarrier (stripping) gas at a flow rate of 10.0 ml/min. The internalcalibrant (o-xylene)-N₂ mixture gas was generated by the permeationmethod.¹⁷ The Pyrex glass cylinder was wrapped with the heating tape anda home-built temperature controller was used to maintain the temperatureat 35° C. The flow rate of the carrier (stripping) gas, which alsoserved as a dilute gas to generate the internal calibrant, wascontrolled by the EPC unit in the SRI GC. Thus, the concentration of theinternal calibrant can be calculated by the usual permeation method.

SPME-GC System for Studying Mass Transfer through a Membrane. In thisstudy, a was designed specifically to facilitate the analysis of themass transfer behaviours of the internal calibrant and the targetanalyte through a membrane. In this system, the membrane module wascovered with a cell base for sample introduction (specific details arepresented in a previous paper).¹⁸ During the experiment, the expectedgas flow rates of the sample stream and carrier (stripping) gas werecontrolled by two EPC control units in the portable model 8610C GC. Thesilicosteel tube (0.75 mm id) was used to connect the individual unitsof the system. The sample gas (at a constant concentration) was obtainedby purging nitrogen through a silanized Pyrex glass cylinder (Φ=3 cm, 13cm length), within which the permeation tube was kept filled with thetarget analytes. This cylinder was also wrapped with heating tape fortemperature control. The exit of the stream was connected with asilanized Pyrex glass sampling vial (Φ=0.5 cm, 10 cm length with 2 cmbranch tube). The vial was sealed with a Teflon lined half-hole-typeThermogreen LB-1 septa (Supelco, Bellefonte, Pa., USA) for SPME samplingand was located close to the membrane module. The actual concentrationof the analyte-enriched stream, prior to entering the membrane module,could be measured in this sampling glass vial via SPME fibre samplingand then analyzed using GC under the conditions described below. A T-teewith a metering valve (Restek Corp., Bellefonte, Pa., USA) was installedin front of a SPME sampling vial to achieve the desired feed velocity.The outlet of the sample side of the membrane module was also connectedto a silanized sampling vial, through which the permeate of the internalcalibrant and the remaining sample were sampled by SPME and analyzed byGC. As constant concentration of the internal calibrant gas was producedby the same method used to generate the target analyte. Theconcentrations of internal calibrant, before and after the membranemodule, and the target analyte permeate concentration were also measuredby SPME through the sampling vial. The gas flowrates in both sides ofmembrane were set up the same value as that used in MESI-portable GCinternal calibrant system and monitored using a calibrated gas flowmeter(Brooks Instrument Division, Emerson Electric Canada, ON, Canada).

GC analyses with SPME were performed using a Varian (Varian Associate,Sunnyvale, Calif.) GC 3400 CX gas chromatograph equipped with a RTX-5capillary column (30 m×0.25 mm i.d.×1.0 μm)(Restek Corp., Bellefonte,Pa., USA), a septum-equipped programmable injector (SPI) with SPME insetand a flame ionization detector (FID). The carrier gas (nitrogen)headpressure was set at 25 psig (˜172 kPa). The initial columntemperature was set at 50° C., held for 1.0 min, and then increased at arate of 20° C. min⁻¹ to 180° C. The temperatures of the injection portand detector were set at 250° C. and 300° C., respectively.

The total mass transfer coefficients of the internal calibrant and thetarget analyte can be calculated individually based on the followingequation:

$\begin{matrix}{h_{total} = \frac{{QH}_{permeant}}{A\left( {H_{feed} - H_{permeant}} \right)}} & (34)\end{matrix}$

Where Q is the gas flow rate in the permeate side, A is the surface areaof the membrane, H_(permeant), and H_(feed) are peak areas for thetarget analyte or internal calibrant between the two sides of themembrane. Using suitable permeate and feed velocities, the permeability(P) of the internal calibrant or target analyte can also be measuredaccording to the following equation:

$\begin{matrix}{P = \frac{\delta_{m}H_{permeant}}{{AQ}\left( {H_{feed} - H_{permeant}} \right)}} & (35)\end{matrix}$Where δ_(m) is the thickness of the membrane.

Materials and Procedures. PDMS membrane (dimethylsilicone membrane,SSP-M100C 0.001, 25 μm thickness) was obtained from Specialty SiliconeProducts Inc. (Ballston Spa, N.Y., USA). All chemicals were ofanalytical grade. Benzene, toluene, ethylbenzene, o-xylene and p-xylenewere purchased from Sigma-Aldrich (Mississauga, ON, Canada). The SPMEholders and 100 μm polydimethylsiloxane (PDMS) fibres were obtained fromSupelco (Oakville, ON, Canada). The fibres were conditioned at 250° C.for 1 hr prior to use.

During the experiments to examine the effect of gas flow rate on thetotal mass transfer coefficient, analytes with different feed velocitieswere obtained by adjusting the metering valve (attached to the T-tee)prior to entrance into the sampling vial, which was located immediatelyupstream of the membrane module. The concentrations were verified bySPME analysis prior to experimental trials were initiated. To study theeffect of the membrane temperature on the extraction efficiency, anOmegalux™ heating tape (Omega, Stamford, Conn., USA) was used to wrapthe membrane module. A thermocouple sensor wire was placed in one holenear the membrane and sealed with asbestos. Both the heating tape andthe thermocouple sensor were connected to an electronic heat controldevice designed and constructed by the Electronics Science Shop at theUniversity of Waterloo. To simplify this experiment, the boundary layersbetween inside and outside membrane were reduced to be neglected levelby using high flowrate (15 cm/s). o-Xylene and p-xylene was used as theinternal calibrant and the sample, respectively.

The determination of the FID response factor was performed by injectinga standard mixture (in methanol) into the same instrument for analysis.For the measurement, the GC conditions, including the carrier gas flowrate, the oven temperature, and the detector temperatures, weremaintained constant to the portable GC-MESI internal calibrant analysisconditions.

Discussion: FIG. 33 presents the total mass transfer coefficients ofo-xylene and p-xylene and the ratios (r) of the mass transfercoefficients of p-xylene to that of o-xylene. As expected, when the feedvelocity of the sample increased, the total mass transfer coefficientsof the internal calibrant and the sample increased simultaneously untila plateau was reached. This illustrated that changing the total masstransfer coefficients of the internal calibrant could reflect theaerodynamic variation of the outside membrane. The observed ratio was0.954±0.009, and likely did not equal one due to the slight differencesof the physicochemical properties of p-xylene and o-xylene.

Effect of Membrane Module Temperatures on the Mass Transfer Coefficient:During on-site (or field) analyses, sometimes the environmentaltemperature cannot be maintained constant and can change from day to dayand from place to place. Thus, the temperature of the membrane modulethat lacks a thermostatic device will vary with temperature of itssurroundings. Membrane extraction efficiency is a function oftemperature. For the correct estimation of chemical concentrations fromthe field MESI and for the development of a suitable calibration method,it is necessary to sufficiently characterize the potential effects oftemperature on the mass transfer through the membrane.

Temperature can change the mass transfer rate by influencing thedistribution constant, the diffusion coefficient, and the boundary layerbetween the membrane and gas. For example, a polyethylene membraneexhibited a permeability increase of about 100% for a 10° C. increase intemperature. PDMS is far superior but still exhibits a decrease inpermeability as the temperature increases. As FIG. 34 illustrates, whenthe temperature of the membrane module was changed from 30 to 100° C.,the mass transfer coefficients of p-xylene and o-xylene varied. Withinambient temperature ranges (30-50° C.), the observed changes were not asgreat. When the temperature continued to increase, the variations ofmass transfer coefficients were no longer insignificant. As noted above,under good convection conditions the mass transfer coefficient throughthe membrane is determined by the permeability (P), and it is atemperature dependent function that obeys the Arrhenius relation:²¹

$\begin{matrix}\begin{matrix}{P = {D \times K}} \\{= {D_{0} \times K_{0} \times \left( {{- \left( {E_{d} + {\Delta\; H_{s}}} \right)} \times \left( {\frac{1}{RT} - \frac{1}{{RT}_{0}}} \right)} \right.}} \\{= {P_{0}{\mathbb{e}}^{\lbrack{- {E_{P}{({\frac{1}{RT} - \frac{1}{{RT}_{0}}})}}}\rbrack}}}\end{matrix} & (36)\end{matrix}$Where D₀ and K₀ represent the diffusion constant and the distributionconstant at some initial temperature, T₀. E_(d) refers to the activationenergy for a diffusion step and the ΔH_(s) are the difference in thesolution heat between the membrane and the sample matrix. In general,E_(d) is larger than zero, which means that the diffusion constantincreases with temperature. Conversely, the ΔH_(s) are less than zero,which means that the distribution constant between the membrane and thesample matrix decreases with the temperature. The overall effect dependson the sum of the effects of K and D during a temperature change.Experimental results indicated that the temperature effect on anincrease in the diffusion coefficient was a trade-off against a decreasein the distribution constant within the observed temperature ranges(30-50° C.). However, the increase of D could not offset the decrease ofK when the temperature of the membrane module was raised beyond 50° C.,and the mass transfer coefficient decreased. On the other hand, withinthe limits of experimental error, the ratio of the mass transfercoefficient of p-xylene to o-xylene was kept constant within the entireexperimental temperature range. This is because o-xylene and p-xyleneare isomers, and they have close values of E_(d) and ΔH_(s), whichexhibit similar trends due to a variation in temperature. This meansthat o-xylene, as an internal calibrant, can also be used to correct thefluctuation of the signal intensity due to a shift in the temperature ofthe membrane module.

FIGS. 33 and 34 demonstrated that effect of flow rates and temperaturevariation can be successfully corrected by introducing the calibrant inthe striping phase and then using ratio between the mass transfer ratescorresponding to the target component (p-xylene) and the calibrant(o-xylene). This indicate suitability of the calibrant in the strippingphase approach for on-site or in-vivo continuous monitoring.

EXAMPLE 11 Hydrophilic Polypyrrole Coatings on Wires, Standards in theExtraction Phase, and Fabrication of Fiber-in-Hypodermic-Needle for Usein a Biological System

An alternative method is described herein, encompassing the preparationof hydrophilic PPY coatings, new samplers based onfiber-in-hypodermic-needle and in-vivo calibration based on the standardon the fiber approach. Use of new metal fiber coating consisting ofmixture of poly(ethylene glycol) and octadecylchlorosilane derivatisedsilica (PEG/C18) coatings is also presented. Methods used herein thatare identical to those in Example 1 are not described.

PPY coating technology is the same as in Example 1, except for theaddition of triethylene glycol to the coating solution. Thismodification results in the preparation of a more hydrophilic and porousPPY; whereas the equilibration time for PPY fibers described in Example1 is 30 min, hydrophilic PPY fibers reach equilibrium in as little as 2min, at a low blood flow rate

FIG. 35 illustrates Diazepam extraction at an intermediate to low flowrate of 50 mL/min. Normal blood flow rate in the cephalic vein of thebeagle dog can be as high as 300 mL/min. Accordingly, 2 min extractiontime ensures that the coating reaches equilibrium.

The reduction of the sampling time to 2 min provides many advantages:convenience of sampling, possibility to monitor drugs with short in-vivolife time, and better correlation with conventional plasma analysis.

An in-vivo sampling device was built inside a stainless steel hypodermicneedle (21 G by 2 in, BD Canada). The 0.005″ wire coated withpolypyrrole was introduced in a suitable hypodermic tube forreinforcement, leaving the coated part outside. The hypodermic tube wasthen introduced inside the needle, sealed with silicone glue, and theupper part was formed into a handle. The new device build inmedical-grade hypodermic needles offers sampling convenience, guaranteedbiocompatibility, minimal exposure to blood, and ease of use inconjunction with the multiwell plate.

In particular, FIG. 36 illustrates how the fiber assemblies 224consisting of the fiber contained in the needle are transported inmultiwell plate 218 to the sampling site in the sealed position toprevent contamination of sterile fibers and to prevent loss of calibrantif present in the coating. FIG. 36 shows multiwell plate 218 havingmultiplicity of wells 220, each of the wells having the narrow part 222,which is able to seal needle 234 of the SPME fiber assembly 224. Each ofthe wells also possesses tapered guiding neck 223 in the upper portionof the well. The SPME module contains fiber 226 containing the coating232 in the lower portion of the fiber located in the needle. The upperend of the fiber contains the attachment module 228, which can be usedto operate the fiber assembly. The SPME fiber assembly also containssealing septa 230, which seals the upper portion of the needle. As FIG.36 indicates when the SPME fiber assemblies are placed inside wells ofthe multiwell plate, the coated part of the fibers are completely sealedin the needle and surrounded well. The sealing plate can be used afterfiber has been exposed to investigated systems to prevent loss ofanalytes or calibrant from the extraction phase. The plate can be placedin the robot to facilitate automated introduction of the fibers to theanalytical instrument to prepare them for parallel desorption inmultiwell plate.

After the experiment is completed the fiber assemblies can bealternatively placed in the holding cover 248 as FIG. 37 illustrates.FIG. 37 illustrates holding cover 248 for the multiwell plates havingmultiple openings 250 consisting of a guiding portion 254 in the upperpart of the well and lower portion 252 to position SPME fiber assembliescorrectly with respect to multiwells containing samples. FIG. 37illustrates the fiber exposed from the needle ready for placement in thewell.

FIG. 38 shows the exposed fiber being placed in the well 262 of themultiwell plate 260 containing desorption solvent or a sample 264. Afterextraction the fibers containing analytes can be desorbed fordeterminations as FIG. 38 shows, or as FIG. 39 illustrates, the fiber226 can be retracted into the needle located in the openings of theholding cover 248 and sealed by the sealing plate 265. In thatarrangement the fibers are sealed in the holding plate. The sealingprevents loss of extracted components of interests, calibrant orcontamination of the fiber. The fibers are then transported tolaboratory for determination in this state. In the laboratory thesealing plate 265 is removed and placed on top of the multiwell plate260 as FIG. 38 illustrates each well containing desorption solvent. Thefibers are lowered to the desorption solvent simultaneously. Thedesorption can be accelerated by agitating the holding cover/multiwellplate system using for example the orbital shaker. Then the desorptionsolvent with desorbed components of interest is introduced to LC/MSinstrumentation or optionally reconstituted to be more compatible withthe mobile phase. Optionally the fibers could be desorbed directly intoanalytical instrument.

Calibration in the in-vivo experiment was done by using two procedures:(1) a comparison with results from an external calibration in wholeblood similar to that shown in FIG. 12; and (2) a standard in the fiberapproach. For the second approach, the fibers (PPY and PEG/C18) werepre-loaded with labeled diazepam (diazepam-D5, Cerilliant, Austin Tex.)by extraction from a solution with 50 ng/mL diazepam-D5 in PBS. Onaverage, PPY fibers were preloaded with 129 ng and PEG/C18 fibers with770 ng diazepam-D5.

The fibers pre-loaded with standard were exposed to the blood flowin-vein for 0.5 min in the case of PPY and 1 min in the case of PEG/C18fibers. Even though the equilibration time is a little longer forPEG/C18, these fibers offer a 5-fold increase in sensitivity because ofhigher capacity of the coating.

FIG. 40, FIG. 41 and FIG. 42 show the results of the use of thefiber-in-hypodermic needle device for the catheter sampling methoddescribed above, for a pharmacokinetic study in dogs. In this case dogswere dosed with 0.5 mg diazepam per kg at time 0:00. Multiple samplingswere performed from a catheter over an interval of 8 hours. Besides theresults obtained with external calibration and standard on the fibercalibration, also shown is a comparison to results obtained by multipleblood draws over the same time period, with conventional samplepreparation and analysis.

FIG. 40 illustrates a pharmacokinetic profile for Diazepam based on anaverage of 9 experiments (3 times on 3 dogs). FIG. 41 illustrates apharmacokinetic profile for Nordiazepam based on an average of 9experiments (3 times on 3 dogs). FIG. 42 illustrates a pharmacokineticprofile for Oxazepam based on an average of 9 experiments (3 times on 3dogs).

These results demonstrate that the device is useful for the applicationdescribed and that the method described produces results in goodagreement with devices and methods using more invasive prior artsampling techniques. PEG/C18 phase showed similar biocompatibilityduring the experiment compared to PPY coating. The calibrant in thefiber approach was investigated for both PPY and PEG/C18 coating forquantification of concentration of components of interests (Diazapam,Oxazapam and Nordiazapam). The internal standard calibration withcalibrant (Diazapan D5) in the fiber coating gave results similar tothat obtained by using external calibration in blood matrix within theRSD of the measurement for these time points.

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto.

1. A solid phase microextraction method of determining the concentrationof a component in a sample, said method comprising adding a calibrant toan extraction phase prior to contact of said extraction phase with saidsample, making said contact between said extraction phase and saidsample for a predetermined extraction time, terminating said contact anddetermining said concentration of said component using amounts of saidcalibrant lost from said extraction phase and said component gained insaid extraction phase after said contact.
 2. The method of claim 1,which includes placement of said extraction phase in a solvent locatedin a well of a multiwell plate after said contact has been terminatedfor desorption of said calibrant and said component from said extractionphase followed by said determination of said amounts.
 3. The method ofclaim 1, wherein said predetermined extraction time is equal to orlonger than the time required to reach equilibrium between saidextraction phase and said sample with respect to said component.
 4. Themethod of claim 1, wherein said predetermined extraction time is shorterthan the time required to reach equilibrium between said extractionphase and said sample with respect to said component.
 5. The method ofclaim 1, wherein the volume of said extraction phase is less than onemilliliter.
 6. The method of claim 1, wherein said extraction phase is apolymer.
 7. The method of claim 1, wherein said extraction phase is in aform of a rod, flat thin membrane or coating.
 8. The method of claim 7,wherein said extraction phase is attached to an external surface of asolid fiber.
 9. The method of claim 7, wherein said extraction phase islocated inside a tube.
 10. The method of claim 1, including anadditional step of placing said extraction phase in an airtight sealingdevice prior to or after making said contact with said sample.
 11. Themethod of claim 1, wherein said calibrant is fully retained in saidextraction phase during said contact of said extraction phase with saidsample.
 12. The method of claim 1, wherein said calibrant is partiallyreleased from said extraction phase during said contact of saidextraction phase with said sample.
 13. The method of claim 1 whereinsaid method is automated and said method includes the step of addingsaid calibrant to said extraction phase prior to said contact of saidextraction phase with said sample.
 14. The method of claim 1, whereinsaid calibrant is an isotopically labeled analogue of said component.